Microbe-Based Products for Enhancing Plant Root and Immune Health

ABSTRACT

Compositions and methods are provided for enhancing plant immunity, health, growth and yields using a combination of microbes and/or their growth by-products. Specifically, the subject invention enhances plant health, growth and/or yields using a combination of a  Trichoderma  spp. fungus and a  Bacillus  spp. bacterium. Specifically, in one embodiment, the subject invention utilizes  Trichoderma harzianum  and  Bacillus amyloliquefaciens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/461,295, filed May 15, 2019; which is a National Stage Application ofInternational Application No. PCT/US2019/031308, filed May 8, 2019;which claims priority to U.S. Provisional Patent Application Ser. No.62/668,316, filed May 8, 2018; and Ser. No. 62/719,758, filed Aug. 20,2018, each of which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

In the agriculture industry, certain common issues continue to hinderthe ability of growers to maximize production yields while keeping costslow. These include, but are not limited to, infections and infestationscaused by bacteria, fungi, nematodes and other pests and pathogens; thehigh costs of chemical fertilizers and herbicides, including theirenvironmental and health impacts; and the difficulty for plants toefficiently absorb nutrients and water from different types of soil.

In citrus production, for example, widespread infection of citrus plantsby pathogens such as those that cause citrus greening disease and citruscanker disease has led to significant hardships for citrus growers. Asmuch as entire crops have been lost to these bacterial infections,leading to a decline in the production, and increase in price, of citrusproducts worldwide.

Citrus greening disease, which is also known is Huanglongbing (HLB) oryellow dragon disease, is an incurable infection caused by theGram-negative bacterium Candidatus Liberibacter asiaticus. This diseasehas caused devastation for millions of acres of citrus crops throughoutthe United States and other parts of the world. Infected trees producefruits that are green, misshapen and bitter, which are unsuitable forsale. The disease is spread by a disease-infected insect, the Asiancitrus psyllid, and has put the future of the world's citrus trees atrisk. HLB lives in, and interferes with the function of, the phloem, orthe plant vascular system that transports sugars to all parts of a tree.Thus, Liberibacter can move to and grow throughout an entire tree,including the roots. Before any expression of foliar symptoms, theinfection typically has already caused significant damage to the rootsystem, causing from 30 to 50% loss in fibrous root density.

Root density continues to gradually decrease as symptoms develop in thecanopy. This is probably due to plugging in the phloem, which restrictsmovement of sugars to the root system. Loss of such a large percentageof the roots greatly reduces the immune health of the tree as well asits ability to absorb nutrients efficiently and to withstand waterstress during extended dry periods. Thus, one of the most crucialcharacteristics for healthy crops is a healthy rhizosphere.

The rhizosphere is the zone of soil wherein a plant's root system growsand absorbs water and nutrients. To supplement soils with certainnutrients, many growers have relied heavily on the use of syntheticchemicals and chemical fertilizers for boosting crop yields andprotecting crops from drought and disease. With reduced uptake capacity,however, when, for example, a plant's root system is compromised due todisease, adding more water and/or nutrients to the soil may not lead toincreased absorption by the root system. Instead, what is applied willflow through the rhizosphere and into the groundwater. As sources ofpollution, responsible use of these substances is an ecological andcommercial imperative. Over-dependence and long-term use of certainchemical fertilizers, pesticides and antibiotics deleteriously alterssoil ecosystems, reduces stress tolerance, increases the prevalence ofresistant pests, and impedes plant growth and vitality.

Efficient nutrient and water absorption in the rhizosphere depends notonly on the amount of water and nutrients present therein, but also uponthe particular microbiome that exists within the soil. Soils containbillions of different microorganisms, which coexist with each other andwith plants to form a complex network of symbiotic relationships.

The optimum combination of microorganisms in a rhizosphere variesbetween the type of plant as well as the type of soil in which it grows.No two plant species or regions will have the same network of microbeswithin a rhizosphere. Thus, while biological agents have the potentialto play an increasingly vital role in crop health and soil remediation,treating a broad range of plant species over many different regionsposes difficulties due to the complexity and specificity of each plant'soptimal rhizospheric microbiome.

The economic costs and the adverse health and environmental impacts ofcurrent methods of crop production continue to burden the sustainabilityof crop-based consumer products. Thus, there is a continuing need forimproved, non-toxic and environmentally-friendly methods of enhancingcrop production at a low cost.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides microbe-based products, as well asmethods of using these microbe-based products in agriculturalapplications. Advantageously, the microbe-based products and methods ofthe subject invention are environmentally-friendly, non-toxic andcost-effective.

In preferred embodiments, the subject invention provides microbe-basedsoil treatment compositions and methods of their use for enhancing thehealth, growth and overall yields of crop plants by, for example,improving the nutrient and moisture retention properties of therhizosphere. Advantageously, the soil treatment compositions of thesubject invention can improve, for example, crop health, as well as cropgrowth and yields, even in situations where one or more of the plants ina crop are infected with a pathogen or where the immune health of thecrop plants is otherwise compromised.

For example, in one embodiment, the subject invention can be used toimprove health, growth and yields of citrus plants infected with, e.g.,Candidatus Liberibacter asiaticus (citrus greening disease) and/orXanthomonas axonopodis (citrus canker disease).

In one embodiment, the subject invention provides soil treatmentcompositions comprising a combination of microorganisms and/or theirgrowth by-products. Also provided are methods of cultivating themicroorganisms and/or growth by-products of the soil treatmentcomposition.

In one embodiment, the soil treatment composition comprises a firstmicroorganism and a second microorganism. More specifically, the firstmicroorganism is a conidia-forming (i.e., spore-forming), non-pathogenicfungal strain, and the second microorganism is a spore-forming,non-pathogenic bacterial strain. Preferably, the composition comprises aTrichoderma spp. fungus and a Bacillus spp. bacterium, although othercombinations are envisioned. In a specific embodiment, the compositioncomprises Trichoderma harzianum and Bacillus amyloliquefaciens.

In one embodiment, the composition can comprise from 1 to 99%Trichoderma by volume and from 99 to 1% Bacillus by volume. In preferredembodiments, the cell count ratio of Trichoderma to Bacillus is about1:4.

In one embodiment, the composition can further comprise one or moreadditional beneficial microorganisms, such as, for example, for example,nitrogen fixers (e.g., Azotobacter vinelandii), potassium mobilizers(e.g., Frateuria aurantia), and others including, for example,Myxococcus xanthus, Pseudomonas chlororaphis, Wickerhamomyces anomalus,Starmerella bombicola, Saccharomyces boulardii, Pichia occidentalis,Pichia kudriavzevii, and/or Meyerozyma guilliermondii.

The species and ratio of microorganisms and other ingredients in thecomposition can be determined according to, for example, the plant beingtreated, the soil type where the plant is growing, the health of theplant at the time of treatment, as well as other factors. Thus, thecomposition can be customizable for any given crop.

The microorganisms of the subject soil treatment compositions can beobtained through cultivation processes ranging from small to largescale. These cultivation processes include, but are not limited to,submerged cultivation/fermentation, solid state fermentation (SSF), andmodifications, hybrids and/or combinations thereof. In preferredembodiments, the microbes are cultivated using SSF or modificationsthereof.

The soil treatment composition can comprise the substrate leftover fromfermentation and/or purified or unpurified growth by-products, such asbiosurfactants, enzymes and/or other metabolites. The microbes can belive or inactive, although in preferred embodiments, the microbes arelive.

The composition is preferably formulated for application to soil, seeds,whole plants, or plant parts (including, but not limited to, roots,tubers, stems, flowers and leaves). In certain embodiments, thecomposition is formulated as, for example, liquid, dust, granules,microgranules, pellets, wettable powder, flowable powder, emulsions,microcapsules, oils, or aerosols.

To improve or stabilize the effects of the composition, it can beblended with suitable adjuvants and then used as such or after dilution,if necessary. In certain embodiments, the composition is formulated as aconcentrated liquid preparation, or as dry powder or dry granules thatcan be mixed with water and other components to form a liquid product.In one embodiment, the composition comprises the substrate, microbes andgrowth by-products, blended together and dried to form powder orgranules.

In one embodiment, the composition can comprise glucose (e.g., in theform of molasses), glycerol, glycerin, and/or other osmoticumsubstances, to promote osmotic pressure during storage and transport ofthe dry product.

In one embodiment, methods are provided for enhancing plant health,growth and/or yields wherein a combination of microorganisms iscontacted with the plant and/or its surrounding environment. The methodcan comprise contacting a soil treatment composition of the subjectinvention, comprising a first microorganism and a second microorganism,and/or a growth by-product of one or both of these microorganisms, withthe plant and/or its surrounding environment. Preferably, the firstmicroorganism is a Trichoderma spp. fungus and the second microorganismis a Bacillus spp. bacterium.

In certain embodiments, the microorganisms of the composition worksynergistically with one another to enhance health, growth and/or yieldsin plants.

In one embodiment, the method can enhance plant health, growth and/oryields by enhancing root health and growth. More specifically, in oneembodiment, the methods can be used to improve the properties of therhizosphere in which a plant's roots are growing, for example, thenutrient and/or moisture retention properties.

Additionally, in one embodiment, the method can be used to inoculate aplant's rhizosphere with one or more beneficial microorganisms. Forexample, in preferred embodiments, the microbes of the soil treatmentcomposition can colonize the rhizosphere and provide multiple benefitsto a plant whose roots are growing therein, including protection andnourishment.

Advantageously, in certain embodiments, the subject methods can be usedto enhance health, growth and/or yields in plants having compromisedimmune health due to an infection from a pathogenic agent or from anenvironmental stressor, such as, for example, drought. Thus, in certainembodiments, the subject methods can also be used for improving theimmune health, or immune response, of plants.

In certain embodiments, the soil treatment composition is contacted witha plant part. In a specific embodiment, the composition is contactedwith one or more roots of the plant. The composition can be applieddirectly to the roots, e.g., by spraying or dunking the roots, and/orindirectly, e.g., by administering the composition to the soil in whichthe plant grows (e.g., the rhizosphere). The composition can be appliedto the seeds of the plant prior to or at the time of planting, or to anyother part of the plant and/or its surrounding environment.

The compositions and methods of the subject invention can be used eitheralone or in combination with other compounds and/or methods forefficiently enhancing plant health, growth and/or yields, and/or forsupplementing the growth of the first and second microbes. For example,in one embodiment, the composition can include and/or can be appliedconcurrently with nutrients and/or micronutrients for enhancing plantand/or microbe growth, such as magnesium, phosphate, nitrogen,potassium, selenium, calcium, sulfur, iron, copper, and zinc; and/or oneor more prebiotics, such as kelp extract, fulvic acid, chitin, humateand/or humic acid. The exact materials and the quantities thereof can bedetermined by a grower or an agricultural scientist having the benefitof the subject disclosure.

The compositions and methods can also be used in combination with othercrop management systems. In one embodiment, the composition canoptionally comprise, or be applied with, natural and/or chemicalpesticides and/or repellants, such as, for example, any known commercialand/or homemade pesticide that is compatible with the combination ofmicroorganisms being applied. In some embodiments, the composition canalso comprise, or be applied with, for example, herbicides, fertilizers,and/or other compatible soil amendments, including commercial productscontaining nutrient sources (e.g., nitrogen-phosphorous-potassium (NPK)and/or micronutrients).

Advantageously, the present invention can be used without releasinglarge quantities of inorganic compounds into the environment.Additionally, the compositions and methods utilize components that arebiodegradable and toxicologically safe. Thus, the present invention canbe used as a “green” soil treatment.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show dry root mass (g) of orange plants in Ft. Basinger,Fla. (A) and grapefruit plants in Ft. Pierce, Fla. (B) treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”) and compared with untreated control (standardgrower's practice) plants.

FIG. 2 shows the dry root mass (g) of grapefruit plants in St. LucieCounty, Fla., treated with a soil treatment composition according to anembodiment of the subject invention (“Rhizolizer™”) and compared withuntreated control (standard grower's practice) plants.

FIGS. 3A-3B show the dry root mass of Hamlin orange trees in HighlandsCounty, Fla. (A) and white grapefruit trees in St. Lucie County, Fla.(B), treated with a soil treatment composition according to anembodiment of the subject invention (“Rhizolizer™”) and compared withuntreated control (standard grower's practice) plants.

FIGS. 4A-4B show the average root weight of Hamlin orange trees in PolkCounty, Fla. (A) and Valencia orange trees in Collier County, Fla. (B),treated with a soil treatment composition according to an embodiment ofthe subject invention (“Rhizolizer™”) and compared with untreatedcontrol (standard grower's practice) plants.

FIGS. 5A-5B show the percent of new shoot growth (A) and average shootcount (B) in young Valencia orange trees in Highlands County, Fla.,treated with a soil treatment composition according to an embodiment ofthe subject invention (“Rhizolizer™”) and compared with untreatedcontrol (standard grower's practice) plants.

FIGS. 6A-6B show increase in canopy density for mature Hamlin orangetrees (A) and young Hamlin orange trees (B) in Highlands County, Fla.,treated with a soil treatment composition according to an embodiment ofthe subject invention (“Rhizolizer™”) and compared with untreatedcontrol (standard grower's practice) plants.

FIG. 7 shows the average caliper change in young Valencia orange treesin Highlands County, Fla., treated with a soil treatment compositionaccording to an embodiment of the subject invention (“Rhizolizer™”) andcompared with untreated control (standard grower's practice) plants.

FIGS. 8A-8B show fruit weight for white grapefruit trees (A) anddiameter for ruby red grapefruit trees (B) in Charlotte County, Fla.,treated with a soil treatment composition according to an embodiment ofthe subject invention (“Rhizolizer™”) and compared with untreatedcontrol (standard grower's practice) plants.

FIGS. 9A-9B show brix measurements for white grapefruits in LucieCounty, Fla. (A) and Charlotte County, Fla. (B), treated with a soiltreatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”) and compared with untreated control (standardgrower's practice) plants.

FIGS. 10A-10B show weight boxes for Hamlin oranges in Lake County, Fla.,treated with a soil treatment composition according to an embodiment ofthe subject invention (“Rhizolizer™”) and compared with untreatedcontrol (standard grower's practice) plants.

FIGS. 11A-11B show total harvest yields in pounds solids for Hamlinoranges in Lake County, Fla., treated with a soil treatment compositionaccording to an embodiment of the subject invention (“Rhizolizer™”) andcompared with untreated control (standard grower's practice) plants.

FIGS. 12A-12B show total harvest yields in pounds solids per box (A) andper acre (B) for Hamlin oranges in Polk County, Fla., treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”) and compared with untreated control (standardgrower's practice) plants.

FIGS. 13A-13B show the fruit yields (tons/acre, y-axis) for tomatoplants treated with a soil treatment composition according to anembodiment of the subject invention (“Rhizolizer™”) compared withuntreated control, commercial check, T. harzianum and B.amyloliquefaciens combined with potato extract, T. harzianum and B.amyloliquefaciens combined with humate and molasses, and Terra Treat.The solid bars represent red fruit yield (harvestable fruit), whereasthe black diamonds mark the total fruit yield for each treatment (A). Aside-by-side comparison of harvestable fruit yield between untreatedcontrol and Rhizolizer™-kelp treatment is shown in (B).

FIGS. 14A-14B show increase in total marketable fruit yields in mT peracre (A) and degrees brix (B) for tomatoes in Fresno County, Calif.,treated with a soil treatment composition according to an embodiment ofthe subject invention (“Rhizolizer™”) and compared with untreatedcontrol (standard grower's practice) plants.

FIGS. 15A-15B shows dry root mass (g) of tomato plants treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), compared with untreated control (standardgrower's practice) plants. 15B shows tomato roots harvested from atomato plant. The roots from the treated plant (bottom image) werevisibly denser than the grower's practice plant (top image).

FIGS. 16A-16B show almond tree trunk diameter measurements when treatedwith a composition according to an embodiment of the subject invention.16A shows the total change in almond tree trunk growth (diameter, mm),where the 6^(th) treatment (Rhizolizer™) produced a change in trunkdiameter of 1.25 mm. Untreated control only measured a 0.96 mm increasein trunk diameter. 16B shows the trunk growth over time of trees treatedwith the Rhizolizer™ treatment.

FIG. 17 shows initial bloom/nut set for almond trees in San JoaquinCounty, Calif., treated with a soil treatment composition according toan embodiment of the subject invention (“Rhizolizer™”), and comparedwith untreated control (standard grower's practice) plants.

FIGS. 18A-18F show trial data comparing root mass increase (A, B),flower count increase (C), fruit count increase (D), melon count andyields per acre (E), and fruit weight and brix values (F) of watermelonplants treated with a composition according to an embodiment of thesubject invention compared with untreated standard grower's practiceplants.

FIG. 19A-19B show trial data comparing flower count (A) and averageharvestable yield (19B) for cantaloupe plants treated with a compositionaccording to an embodiment of the subject invention compared withuntreated standard grower's practice plants.

FIGS. 20A-20B show potato plant emergence in Imperial County, Calif. foryellow rose russet potatoes planted 5 weeks after planting (A) and multicultivars 6 weeks after planting (B). The potatoes were treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), and compared with untreated control (standardgrower's practice) plants.

FIGS. 21A-21B shows potato yields Reveille Russet Fresh Market Bakingpotatoes in Hartley County, Tex. (A) and Russet Burbank potatoes inWalworth County, Wis. (B). The potatoes were treated with a soiltreatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), and compared with untreated control (standardgrower's practice) plants.

FIGS. 22A-22B show potato harvest quality for Russet Burbank potatoes(A) and yield by grade for Colomba potatoes (B). The potatoes weretreated with a soil treatment composition according to an embodiment ofthe subject invention (“Rhizolizer™”), and compared with untreatedcontrol (standard grower's practice) plants.

FIGS. 23A-23C show increase in fruit size for strawberries treated witha soil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), compared with untreated control (standardgrower's practice) plants. Fruit diameter (A-B) and fruit weight (C)were measured.

FIG. 24 shows increase in Brix Rating for strawberries treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), compared with untreated control (standardgrower's practice) plants.

FIG. 25 shows increase in canopy width for strawberries treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), compared with untreated control (standardgrower's practice) plants.

FIG. 26 shows increase in root mass for strawberries treated with a soiltreatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), compared with untreated control (standardgrower's practice) plants.

FIG. 27 shows increase in fruit count for strawberries treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), compared with untreated control (standardgrower's practice) plants.

FIGS. 28A-28B show increase in mid-season fruit yields for strawberries.Average flats/acre (A) and total flats (B) were measured. Thestrawberries were treated with a soil treatment composition according toan embodiment of the subject invention (“Rhizolizer™”), and comparedwith untreated control (standard grower's practice) plants.

FIG. 29 shows increase in root weight for corn plants treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), either once at planting, once at mid-season,or twice (once at planting and once at mid-season). The three treatmentgroups were compared with untreated control (standard grower's practice)plants.

FIG. 30 shows increase in plant vigor for corn plants treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), either once at planting, once at mid-season,or twice (once at planting and once at mid-season). The three treatmentgroups were compared with untreated control (standard grower's practice)plants.

FIG. 31 shows increase in yield for corn plants treated with a soiltreatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), either once at planting, once at mid-season,or twice (once at planting and once at mid-season). The three treatmentgroups were compared with untreated control (standard grower's practice)plants.

FIG. 32 shows increase in projected yield for two groups of cottonplants treated with a soil treatment composition according to anembodiment of the subject invention (“Rhizolizer™”), compared withuntreated control (standard grower's practice) plants.

FIG. 33 shows increase in average chlorophyll (top left), average leafwidth (top right), and average leaf length (bottom) for tobacco plantstreated with a soil treatment composition according to an embodiment ofthe subject invention (“Rhizolizer™”), compared with untreated control(standard grower's practice) plants.

FIG. 34 shows increase in projected yield for peanut plants treated witha soil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), compared with untreated control (standardgrower's practice) plants.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides microbe-based products, as well asmethods of using these microbe-based products in agriculturalapplications. Advantageously, the microbe-based products and methods ofthe subject invention are environmentally-friendly, non-toxic andcost-effective.

In preferred embodiments, the subject invention provides microbe-basedsoil treatment compositions and methods of their use for enhancing thehealth, growth and overall yields of crop plants by, for example,improving the nutrient and moisture retention properties of therhizosphere. Advantageously, the soil treatment compositions of thesubject invention can improve, for example, crop health, as well as cropgrowth and yields, even in situations where one or more of the plants ina crop are infected with a pathogen or where the immune health of thecrop plants is otherwise compromised.

Selected Definitions

The subject invention utilizes “microbe-based compositions,” meaning acomposition that comprises components that were produced as the resultof the growth of microorganisms or other cell cultures. Thus, themicrobe-based composition may comprise the microbes themselves and/orby-products of microbial growth. The microbes may be in a vegetativestate, in spore or conidia form, in hyphae form, in any other form ofpropagule, or a mixture of these. The microbes may be planktonic or in abiofilm form, or a mixture of both. The by-products of growth may be,for example, metabolites, cell membrane components, expressed proteins,and/or other cellular components. The microbes may be intact or lysed.In preferred embodiments, the microbes are present, with growth mediumin which they were grown, in the microbe-based composition. The microbesmay be present at, for example, a concentration of at least 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹² or 1×10¹³ ormore CFU per gram or per ml of the composition.

The subject invention further provides “microbe-based products,” whichare products that are to be applied in practice to achieve a desiredresult. The microbe-based product can be simply the microbe-basedcomposition harvested from the microbe cultivation process.Alternatively, the microbe-based product may comprise furtheringredients that have been added. These additional ingredients caninclude, for example, stabilizers, buffers, appropriate carriers, suchas water, salt solutions, or any other appropriate carrier, addednutrients to support further microbial growth, non-nutrient growthenhancers and/or agents that facilitate tracking of the microbes and/orthe composition in the environment to which it is applied. Themicrobe-based product may also comprise mixtures of microbe-basedcompositions. The microbe-based product may also comprise one or morecomponents of a microbe-based composition that have been processed insome way such as, but not limited to, filtering, centrifugation, lysing,drying, purification and the like.

As used herein, “harvested” in the context of fermentation of amicrobe-based composition refers to removing some or all of themicrobe-based composition from a growth vessel.

As used herein, a “biofilm” is a complex aggregate of microorganisms,wherein the cells adhere to each other and/or to surfaces. In someembodiments, the cells secrete a polysaccharide barrier that surroundsthe entire aggregate. The cells in biofilms are physiologically distinctfrom planktonic cells of the same organism, which are single cells thatcan float or swim in liquid medium.

As used herein, an “isolated” or “purified” compound is substantiallyfree of other compounds, such as cellular material, with which it isassociated in nature. A purified or isolated polynucleotide (ribonucleicacid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes orsequences that flank it in its naturally-occurring state. A purified orisolated polypeptide is free of the amino acids or sequences that flankit in its naturally-occurring state. “Isolated” in the context of amicrobial strain means that the strain is removed from the environmentin which it exists in nature. Thus, the isolated strain may exist as,for example, a biologically pure culture, or as spores (or other formsof the strain) in association with a carrier.

As used herein, a “biologically pure culture” is a culture that has beenisolated from materials with which it is associated in nature. In apreferred embodiment, the culture has been isolated from all otherliving cells. In further preferred embodiments, the biologically pureculture has advantageous characteristics compared to a culture of thesame microbe as it exists in nature. The advantageous characteristicscan be, for example, enhanced production of one or more growthby-products.

In certain embodiments, purified compounds are at least 60% by weightthe compound of interest. Preferably, the preparation is at least 75%,more preferably at least 90%, and most preferably at least 99%, byweight the compound of interest. For example, a purified compound is onethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w)of the desired compound by weight. Purity is measured by any appropriatestandard method, for example, by column chromatography, thin layerchromatography, or high-performance liquid chromatography (HPLC)analysis.

A “metabolite” refers to any substance produced by metabolism (e.g., agrowth by-product) or a substance necessary for taking part in aparticular metabolic process. Examples of metabolites include, but arenot limited to, biosurfactants, biopolymers, enzymes, acids, solvents,alcohols, proteins, vitamins, minerals, microelements, and amino acids.

As used herein, “modulate” means to cause an alteration (e.g., increaseor decrease).

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 20 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, as well as all intervening decimal values betweenthe aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nestedsub-ranges” that extend from either end point of the range arespecifically contemplated. For example, a nested sub-range of anexemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 inthe other direction.

As used herein, “reduce” refers to a negative alteration, and the term“increase” refers to a positive alteration, each of at least 1%, 5%,10%, 25%, 50%, 75%, or 100%.

As used herein, “reference” refers to a standard or control condition.

As used herein, “surfactant” refers to a compound that lowers thesurface tension (or interfacial tension) between two liquids or betweena liquid and a solid. Surfactants act as, e.g., detergents, wettingagents, emulsifiers, foaming agents, and dispersants. A “biosurfactant”is a surfactant produced by a living organism.

As used herein, “agriculture” means the cultivation and breeding ofplants, algae and/or fungi for food, fiber, biofuel, medicines,cosmetics, supplements, ornamental purposes and other uses. According tothe subject invention, agriculture can also include horticulture,landscaping, gardening, plant conservation, orcharding andarboriculture. Further included in agriculture is the care, monitoringand maintenance of soil.

As used herein, “enhancing” means improving or increasing. For example,enhanced plant health means improving the plant's ability grow andthrive, which includes the plant's ability to ward off pests and/ordiseases, and to survive environmental stressors, such as droughtsand/or overwatering. Enhanced plant growth means increasing the sizeand/or mass of a plant, and/or improving the ability of the plant toreach a desired size and/or mass. Enhanced yields mean improving thequantity, quality (e.g., taste, texture) and/or size of end productsproduced by a plant.

As used herein “preventing” or “prevention” of a situation or occurrencemeans delaying, inhibiting, suppressing, forestalling, and/or minimizingthe onset, extensiveness or progression of the situation or occurrence.Prevention can include, but does not require, indefinite, absolute orcomplete prevention, meaning the sign or symptom may still develop at alater time. Prevention can include reducing the severity of the onset ofsuch a disease, condition or disorder, and/or inhibiting the progressionof the condition or disorder to a more severe condition or disorder.

As used herein, the term “control” used in reference to a pest meanskilling, disabling, immobilizing, or reducing population numbers of apest, or otherwise rendering the pest substantially incapable of causingharm.

As used herein, a “pest” is any organism, other than a human, that isdestructive, deleterious and/or detrimental to humans or human concerns(e.g., agriculture, horticulture). In some, but not all instances, apest may be a pathogenic organism. Pests may cause or be a vector forinfections, infestations and/or disease, or they may simply feed on orcause other physical harm to living tissue. Pests may be single- ormulti-cellular organisms, including but not limited to, viruses, fungi,bacteria, parasites, and/or nematodes.

As used herein, a “soil amendment” or a “soil conditioner” is anycompound, material, or combination of compounds or materials that areadded into soil to enhance the physical properties of the soil. Soilamendments can include organic and inorganic matter, and can furtherinclude, for example, fertilizers, pesticides and/or herbicides.Nutrient-rich, well-draining soil is essential for the growth and healthof plants, and thus, soil amendments can be used for enhancing thegrowth and health of plants by altering the nutrient and moisturecontent of soil. Soil amendments can also be used for improving manydifferent qualities of soil, including but not limited to, soilstructure (e.g., preventing compaction); improving the nutrientconcentration and storage capabilities; improving water retention in drysoils; and improving drainage in waterlogged soils.

As used herein, “environmental stressor” refers to an abiotic, ornon-living, condition that has a negative impact on a living organism ina specific environment. The environmental stressor must influence theenvironment beyond its normal range of variation to adversely affect thepopulation performance or individual physiology of the organism in asignificant way. Examples of environmental stressors include, but arenot limited to, drought, extreme temperatures, flood, high winds,natural disasters, soil pH changes, high radiation, compaction of soil,pollution, and others.

The transitional term “comprising,” which is synonymous with“including,” or “containing,” is inclusive or open-ended and does notexclude additional, unrecited elements or method steps. By contrast, thetransitional phrase “consisting of” excludes any element, step, oringredient not specified in the claim. The transitional phrase“consisting essentially of” limits the scope of a claim to the specifiedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. Use of the term“comprising” contemplates other embodiments that “consist” or “consistessentially of” the recited component(s).

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a,” “and” and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

All references cited herein are hereby incorporated by reference intheir entirety.

Soil Treatment Compositions

In one embodiment, the subject invention provides soil treatmentcompositions comprising a combination of microorganisms and/or theirgrowth by-products. The soil treatment composition can be used toenhance plant health, growth and/or yields, and in some embodiments,even in plants that have been infected by a pathogen or disease. Morespecifically, the subject compositions can be used to enhance the rootgrowth and health, and/or to enhance the immune health of a plant. Incertain embodiments, the soil treatment composition can also be used toinoculate plant roots with one or more beneficial microorganisms.

Advantageously, the microbe-based compositions according to the subjectinvention are non-toxic and can be applied in high concentrationswithout causing irritation to, for example, the skin or digestive tractof a human or other non-pest animal. Thus, the subject invention isparticularly useful where application of the microbe-based compositionsoccurs in the presence of living organisms, such as growers andlivestock.

In one embodiment, the soil treatment composition can comprise a firstmicroorganism, which is preferably a conidia-forming (spore-forming)fungal strain, and a second microorganism, which is preferably aspore-forming bacterial strain. Preferably, the first microorganism is aTrichoderma spp. fungus and the second microorganism is a spore-formingBacillus spp. bacterium, although other combinations are envisioned. Incertain embodiments, the composition comprises Trichoderma harzianum andBacillus amyloliquefaciens. In a specific embodiment, the strain of B.amyloliquefaciens is B. amyloliquefaciens subsp. locus.

A culture of Bacillus amyloliquefaciens subsp locus microbe usefulaccording to the subject invention, has been deposited with the NorthernRegional Research Laboratory (NRRL) Culture Collection, USDA, ARS,NCAUR, MPM, 1815 N. University St., PEORIA, Ill. 61604, USA. Themicroorganism was deposited on Feb. 26, 2020 and was assigned accessionnumber NRRL B-67928 by the depository.

A culture of Wickerhamomyces anomalus microbe useful according to thesubject invention, has been deposited with the Northern RegionalResearch Laboratory (NRRL) Culture Collection, USDA, ARS, NCAUR, MPM,1815 N. University St., PEORIA, Ill. 61604, USA. The microorgainsm wasdeposited on May 6, 2021 and was assigned accession number NRRL Y-68030by the depository.

A culture of Bacillus subtilis B4 microbe useful according to thesubject invention, has been deposited with the Northern RegionalResearch Laboratory (NRRL) Culture Collection, USDA, ARS, NCAUR, MPM,1815 N. University St., PEORIA, Ill. 61604, USA.

The microorgainsm was deposited on May 6, 2021 and was assignedaccession number NRRL B-68031 by the depository.

Each of the subject cultures has been deposited under conditions thatassure that access to the culture will be available during the pendencyof this patent application to one determined by the Commissioner ofPatents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35U.S.C 122. Each deposit is available as required by foreign patent lawsin countries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., each deposit will be stored withall the care necessary to keep it viable and uncontaminated for a periodof at least five years after the most recent request for the furnishingof a sample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent that may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of each ofthe subject culture deposits will be irrevocably removed upon thegranting of a patent disclosing it.

In one embodiment, the composition can comprise from 1 to 99%Trichoderma by weight and from 99 to 1% Bacillus by weight. In someembodiments, the cell count ratio of Trichoderma to Bacillus is about1:9 to about 9:1, about 1:8 to about 8:1, about 1:7 to about 7:1, about1:6 to about 6:1, about 1:5 to about 5:1 or about 1:4 to about 4:1.

In one embodiment, the microorganisms of the subject compositioncomprise about 5 to 20% of the total composition by weight, or about 8to 15%, or about 10 to 12%. In one embodiment, the composition comprisesabout 1×10⁶ to 1×10¹², 1×10⁷ to 1×10¹¹, 1×10⁸ to 1×10¹⁰, or 1×10⁹ CFU/mlof Trichoderma. In one specific embodiment, the composition comprisesabout 1×10⁶ to 1×10¹², 1×10⁷ to 1×10¹¹, 1×10⁸ to 1×10¹⁰, or 1×10⁹ CFU/mlof Bacillus.

In some embodiments, the composition can further comprise one or moreadditional microbes. In one embodiment, the additional microbes cancomprise one or more of, for example, a mycobacterium and/or other typeof bacteria, a yeast and/or a fungus. In an exemplary embodiment, amycobacterium is included, wherein the mycobacterium is Myxococcusxanthus.

In certain embodiments, the additional microbes are capable of fixing,solubilizing and/or mobilizing nitrogen, potassium, phosphorous (orphosphate) and/or other micronutrients in soil. In one embodiment, anitrogen-fixing bacteria can be included, such as, for example,Azotobacter vinelandii. In another embodiment, a potassium-mobilizingbacteria can be included, such as, for example, Frateuria aurantia.

Other additional microbes can include, for example, Pseudomonaschlororaphis, Wickerhamomyces anomalus, Starmerella bombicola,Saccharomyces boulardii, Pichia occidentalis, Pichia kudriavzevii,and/or Meyerozyma guilliermondii.

In a specific embodiment, the one or more additional microbes are addedat a concentration of 1×10⁸ to 1×10¹¹, or 1×10⁹ to 1×10¹⁰ CFU/ml each.

The species and ratio of microorganisms and other ingredients in thecomposition can be customized according to, for example, the plant beingtreated, the soil type where the plant is growing, the health of theplant at the time of treatment, as well as other factors.

The microbes and microbe-based compositions of the subject inventionhave a number of beneficial properties that are useful for enhancingplant health, growth and/or yields. For example, the compositions cancomprise products resulting from the growth of the microorganisms, suchas biosurfactants, proteins and/or enzymes, either in purified or crudeform.

In one embodiment, the microorganisms of the subject composition arecapable of producing a biosurfactant. In another embodiment,biosurfactants can be produced separately by other microorganisms andadded to the composition, either in purified form or in crude form.Crude form biosurfactants can comprise, for example, biosurfactants andother products of cellular growth in the leftover fermentation mediumresulting from cultivation of a biosurfactant-producing microbe. Thiscrude form biosurfactant composition can comprise from about 0.001% toabout 90%, about 25% to about 75%, about 30% to about 70%, about 35% toabout 65%, about 40% to about 60%, about 45% to about 55%, or about 50%pure biosurfactant.

Biosurfactants form an important class of secondary metabolites producedby a variety of microorganisms such as bacteria, fungi, and yeasts. Asamphiphilic molecules, microbial biosurfactants reduce the surface andinterfacial tensions between the molecules of liquids, solids, andgases. Furthermore, the biosurfactants according to the subjectinvention are biodegradable, have low toxicity, are effective insolubilizing and degrading insoluble compounds in soil and can beproduced using low cost and renewable resources. They can inhibitadhesion of undesirable microorganisms to a variety of surfaces, preventthe formation of biofilms, and can have powerful emulsifying anddemulsifying properties. Furthermore, the biosurfactants can also beused to improve wettability and to achieve even solubilization and/ordistribution of fertilizers, nutrients, and water in the soil.

Biosurfactants according to the subject methods can be selected from,for example, low molecular weight glycolipids (e.g., sophorolipids,cellobiose lipids, rhamnolipids, mannosylerythritol lipids and trehaloselipids), lipopeptides (e.g., surfactin, iturin, fengycin, arthrofactinand lichenysin), flavolipids, phospholipids (e.g., cardiolipins), andhigh molecular weight polymers such as lipoproteins,lipopolysaccharide-protein complexes, and polysaccharide-protein-fattyacid complexes.

The composition can comprise one or more biosurfactants at aconcentration of 0.001% to 10%, 0.01% to 5%, 0.05% to 2%, and/or from0.1% to 1%. Advantageously, in accordance with the subject invention,the soil treatment composition may comprise the medium in which each ofthe microorganism were grown.

The composition may be, for example, at least, by weight, 1%, 5%, 10%,25%, 50%, 75%, or 100% growth medium.

The fermentation medium can contain a live and/or an inactive culture,purified or crude form growth by-products, such as biosurfactants,enzymes, and/or other metabolites, and/or any residual nutrients. Theamount of biomass in the composition, by weight, may be, for example,anywhere from about 0.01% to 100%, about 1% to 90%, about 5% to about80%, or about 10% to about 75%.

The product of fermentation may be used directly, with or withoutextraction or purification. If desired, extraction and purification canbe easily achieved using standard extraction and/or purification methodsor techniques described in the literature.

The microorganisms in the soil treatment composition may be in an activeor inactive form, or in the form of vegetative cells, reproductivespores, mycelia, hyphae, conidia or any other form of microbialpropagule. The composition may also contain a combination of any ofthese microbial forms.

In one embodiment, different species of microorganism are grownseparately and then mixed together to produce the soil treatmentcomposition. In one embodiment, microorganisms can be co-cultivated, forexample, B. amyloliquefaciens and M. xanthus.

In one embodiment, the composition is preferably formulated forapplication to soil, seeds, whole plants, or plant parts (including, butnot limited to, roots, tubers, stems, flowers and leaves). In certainembodiments, the composition is formulated as, for example, liquid,dust, granules, microgranules, pellets, wettable powder, flowablepowder, emulsions, microcapsules, oils, or aerosols.

To improve or stabilize the effects of the composition, it can beblended with suitable adjuvants and then used as such or after dilution,if necessary. In preferred embodiments, the composition is formulated asa liquid, a concentrated liquid, or as dry powder or granules that canbe mixed with water and other components to form a liquid product.

In one embodiment, the composition can comprise glucose (e.g., in theform of molasses), glycerol and/or glycerin, as, or in addition to, anosmoticum substance, to promote osmotic pressure during storage andtransport of the dry product.

The compositions can be used either alone or in combination with othercompounds and/or methods for efficiently enhancing plant health, growthand/or yields, and/or for supplementing the growth of the first andsecond microbes. For example, in one embodiment, the composition caninclude and/or can be applied concurrently with nutrients and/ormicronutrients for enhancing plant and/or microbe growth, such asmagnesium, phosphate, nitrogen, potassium, selenium, calcium, sulfur,iron, copper, and zinc; and/or one or more prebiotics, such as kelpextract, fulvic acid, chitin, humate and/or humic acid. The exactmaterials and the quantities thereof can be determined by a grower or anagricultural scientist having the benefit of the subject disclosure.

The compositions can also be used in combination with other agriculturalcompounds and/or crop management systems. In one embodiment, thecomposition can optionally comprise, or be applied with, for example,natural and/or chemical pesticides, repellants, herbicides, fertilizers,water treatments, non-ionic surfactants and/or soil amendments.Preferably, however, the composition does not comprise and/or is notused with benomyl, dodecyl dimethyl ammonium chloride, hydrogendioxide/peroxyacetic acid, imazilil, propiconazole, tebuconazole, ortriflumizole.

If the composition is mixed with compatible chemical additives, thechemicals are preferably diluted with water prior to addition of thesubject composition.

Further components can be added to the composition, for example,buffering agents, carriers, other microbe-based compositions produced atthe same or different facility, viscosity modifiers, preservatives,nutrients for microbe growth, tracking agents, biocides, other microbes,surfactants, emulsifying agents, lubricants, solubility controllingagents, pH adjusting agents, preservatives, stabilizers and ultra-violetlight resistant agents.

The pH of the microbe-based composition should be suitable for themicroorganism of interest. In a preferred embodiment, the pH of thecomposition is about 3.5 to 7.0, about 4.0 to 6.5, or about 5.0.

Optionally, the composition can be stored prior to use. The storage timeis preferably short. Thus, the storage time may be less than 60 days, 45days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2days, 1 day, or 12 hours. In a preferred embodiment, if live cells arepresent in the product, the product is stored at a cool temperature suchas, for example, less than 20° C., 15° C., 10° C., or 5° C.

The microbe-based compositions may be used without furtherstabilization, preservation, and storage, however. Advantageously,direct usage of these microbe-based compositions preserves a highviability of the microorganisms, reduces the possibility ofcontamination from foreign agents and undesirable microorganisms, andmaintains the activity of the by-products of microbial growth.

In other embodiments, the composition (microbes, growth medium, ormicrobes and medium) can be placed in containers of appropriate size,taking into consideration, for example, the intended use, thecontemplated method of application, the size of the fermentation vessel,and any mode of transportation from microbe growth facility to thelocation of use. Thus, the containers into which the microbe-basedcomposition is placed may be, for example, from 1 pint to 1,000 gallonsor more. In certain embodiments the containers are 1 gallon, 2 gallons,5 gallons, 25 gallons, or larger.

Growth of Microbes According to the Subject Invention

The subject invention utilizes methods for cultivation of microorganismsand production of microbial metabolites and/or other by-products ofmicrobial growth. The subject invention further utilizes cultivationprocesses that are suitable for cultivation of microorganisms andproduction of microbial metabolites on a desired scale. Thesecultivation processes include, but are not limited to, submergedcultivation/fermentation, solid state fermentation (SSF), andmodifications, hybrids and/or combinations thereof.

As used herein “fermentation” refers to cultivation or growth of cellsunder controlled conditions. The growth could be aerobic or anaerobic.In preferred embodiments, the microorganisms are grown using SSF and/ormodified versions thereof.

In one embodiment, the subject invention provides materials and methodsfor the production of biomass (e.g., viable cellular material),extracellular metabolites (e.g. small molecules and excreted proteins),residual nutrients and/or intracellular components (e.g. enzymes andother proteins).

The microbe growth vessel used according to the subject invention can beany fermenter or cultivation reactor for industrial use. In oneembodiment, the vessel may have functional controls/sensors or may beconnected to functional controls/sensors to measure important factors inthe cultivation process, such as pH, oxygen, pressure, temperature,humidity, microbial density and/or metabolite concentration.

In a further embodiment, the vessel may also be able to monitor thegrowth of microorganisms inside the vessel (e.g., measurement of cellnumber and growth phases). Alternatively, a daily sample may be takenfrom the vessel and subjected to enumeration by techniques known in theart, such as dilution plating technique. Dilution plating is a simpletechnique used to estimate the number of organisms in a sample. Thetechnique can also provide an index by which different environments ortreatments can be compared.

In one embodiment, the method includes supplementing the cultivationwith a nitrogen source. The nitrogen source can be, for example,potassium nitrate, ammonium nitrate ammonium sulfate, ammoniumphosphate, ammonia, urea, and/or ammonium chloride. These nitrogensources may be used independently or in a combination of two or more.

The method can provide oxygenation to the growing culture. Oneembodiment utilizes slow motion of air to remove low-oxygen containingair and introduce oxygenated air. In the case of submerged fermentation,the oxygenated air may be ambient air supplemented daily throughmechanisms including impellers for mechanical agitation of liquid, andair spargers for supplying bubbles of gas to liquid for dissolution ofoxygen into the liquid.

The method can further comprise supplementing the cultivation with acarbon source. The carbon source is typically a carbohydrate, such asglucose, sucrose, lactose, fructose, trehalose, mannose, mannitol,and/or maltose; organic acids such as acetic acid, fumaric acid, citricacid, propionic acid, malic acid, malonic acid, and/or pyruvic acid;alcohols such as ethanol, propanol, butanol, pentanol, hexanol,isobutanol, and/or glycerol; fats and oils such as soybean oil, canolaoil, rice bran oil, olive oil, corn oil, sesame oil, and/or linseed oil;etc. These carbon sources may be used independently or in a combinationof two or more.

In one embodiment, growth factors and trace nutrients for microorganismsare included in the medium. This is particularly preferred when growingmicrobes that are incapable of producing all of the vitamins theyrequire. Inorganic nutrients, including trace elements such as iron,zinc, copper, manganese, molybdenum and/or cobalt may also be includedin the medium. Furthermore, sources of vitamins, essential amino acids,and microelements can be included, for example, in the form of flours ormeals, such as corn flour, or in the form of extracts, such as yeastextract, potato extract, beef extract, soybean extract, banana peelextract, and the like, or in purified forms. Amino acids such as, forexample, those useful for biosynthesis of proteins, can also beincluded.

In one embodiment, inorganic salts may also be included. Usableinorganic salts can be potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate,magnesium chloride, iron sulfate, iron chloride, manganese sulfate,manganese chloride, zinc sulfate, lead chloride, copper sulfate, calciumchloride, sodium chloride, calcium carbonate, and/or sodium carbonate.These inorganic salts may be used independently or in a combination oftwo or more.

In some embodiments, the method for cultivation may further compriseadding additional acids and/or antimicrobials in the medium before,and/or during the cultivation process. Antimicrobial agents orantibiotics are used for protecting the culture against contamination.

Additionally, antifoaming agents may also be added to prevent theformation and/or accumulation of foam during submerged cultivation.

The pH of the mixture should be suitable for the microorganism ofinterest. Buffers, and pH regulators, such as carbonates and phosphates,may be used to stabilize pH near a preferred value. When metal ions arepresent in high concentrations, use of a chelating agent in the mediummay be necessary.

The microbes can be grown in planktonic form or as biofilm. In the caseof biofilm, the vessel may have within it a substrate upon which themicrobes can be grown in a biofilm state. The system may also have, forexample, the capacity to apply stimuli (such as shear stress) thatencourages and/or improves the biofilm growth characteristics.

In one embodiment, the method for cultivation of microorganisms iscarried out at about 5° to about 100° C., preferably, 15 to 60° C., morepreferably, 25 to 50° C. In a further embodiment, the cultivation may becarried out continuously at a constant temperature. In anotherembodiment, the cultivation may be subject to changing temperatures.

In one embodiment, the equipment used in the method and cultivationprocess is sterile. The cultivation equipment such as the reactor/vesselmay be separated from, but connected to, a sterilizing unit, e.g., anautoclave. The cultivation equipment may also have a sterilizing unitthat sterilizes in situ before starting the inoculation. Air can besterilized by methods know in the art. For example, the ambient air canpass through at least one filter before being introduced into thevessel. In other embodiments, the medium may be pasteurized or,optionally, no heat at all added, where the use of low water activityand low pH may be exploited to control undesirable bacterial growth.

In one embodiment, the subject invention further provides a method forproducing microbial metabolites such as, for example, biosurfactants,enzymes, proteins, ethanol, lactic acid, beta-glucan, peptides,metabolic intermediates, polyunsaturated fatty acid, and lipids, bycultivating a microbe strain of the subject invention under conditionsappropriate for growth and metabolite production; and, optionally,purifying the metabolite. The metabolite content produced by the methodcan be, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

The microbial growth by-product produced by microorganisms of interestmay be retained in the microorganisms or secreted into the growthmedium. The medium may contain compounds that stabilize the activity ofmicrobial growth by-product.

The biomass content of the fermentation medium may be, for example, from5 g/l to 180 g/l or more, or from 10 g/l to 150 g/l.

The cell concentration may be, for example, at least 1×10⁶ to 1×10¹²,1×10⁷ to 1×10¹¹, 1×10⁸ to 1×10¹⁰, or 1×10⁹ CFU/ml.

The method and equipment for cultivation of microorganisms andproduction of the microbial by-products can be performed in a batch, aquasi-continuous process, or a continuous process.

In one embodiment, all of the microbial cultivation composition isremoved upon the completion of the cultivation (e.g., upon, for example,achieving a desired cell density, or density of a specified metabolite).In this batch procedure, an entirely new batch is initiated uponharvesting of the first batch.

In another embodiment, only a portion of the fermentation product isremoved at any one time. In this embodiment, biomass with viable cells,spores, conidia, hyphae and/or mycelia remains in the vessel as aninoculant for a new cultivation batch. The composition that is removedcan be a cell-free medium or contain cells, spores, or otherreproductive propagules, and/or a combination of thereof. In thismanner, a quasi-continuous system is created.

Advantageously, the method does not require complicated equipment orhigh energy consumption. The microorganisms of interest can becultivated at small or large scale on site and utilized, even beingstill-mixed with their media.

Advantageously, the microbe-based products can be produced in remotelocations.

The microbe growth facilities may operate off the grid by utilizing, forexample, solar, wind and/or hydroelectric power.

Microbial Strains

The microorganisms useful according to the subject invention can be, forexample, non-plant-pathogenic strains of bacteria, yeast and/or fungi.These microorganisms may be natural, or genetically modifiedmicroorganisms. For example, the microorganisms may be transformed withspecific genes to exhibit specific characteristics. The microorganismsmay also be mutants of a desired strain. As used herein, “mutant” meansa strain, genetic variant or subtype of a reference microorganism,wherein the mutant has one or more genetic variations (e.g., a pointmutation, missense mutation, nonsense mutation, deletion, duplication,frameshift mutation or repeat expansion) as compared to the referencemicroorganism. Procedures for making mutants are well known in themicrobiological art.

For example, UV mutagenesis and nitrosoguanidine are used extensivelytoward this end. In one embodiment, the microorganism is a yeast orfungus. Yeast and fungus species suitable for use according to thecurrent invention, include Aureobasidium (e.g., A. pullulans),Blakeslea, Candida (e.g., C. apicola, C. bombicola, C. nodaensis),Cryptococcus, Debaryomyces (e.g., D. hansenii), Entomophthora,Hanseniaspora, (e.g., H. uvarum), Hansenula, Issatchenkia, Kluyveromyces(e.g., K. phaffii), Mortierella, Mycorrhiza, Penicillium, Phycomyces,Pichia (e.g., P. anomala, P. guilliermondii, P. occidentalis, P.kudriavzevii), Pleurotus spp. (e.g., P. ostreatus), Pseudozyma (e.g., P.aphidis), Saccharomyces (e.g., S. boulardii sequela, S. cerevisiae, S.torula), Starmerella (e.g., S. bombicola), Torulopsis, Trichoderma(e.g., T. reesei, T. harzianum, T. hamatum, T. viride), Ustilago (e.g.,U. maydis), Wickerhamomyces (e.g., W. anomalus), Williopsis (e.g., W.mrakii), Zygosaccharomyces (e.g., Z. bailii), and others.

In a preferred embodiment, the microorganism is a spore-producingTrichoderma spp. fungus. In a specific preferred embodiment, themicroorganism is Trichoderma harzianum.

Certain species of Trichoderma are useful when added to soil, where theycan multiply and grow in close association with plants' roots. They arecapable of partially protecting the roots from invasion by other plantpathogenic fungi and other microbial and animal pests, in addition tohelping to stimulate plant growth.

Trichoderma can establish strong and long-lasting colonization of rootsurfaces, penetrating into the epidermis and shallow subsurface cells.These root-microorganism associations cause substantial changes to theplant proteome and metabolism. They produce and/or release a variety ofcompounds that induce localized or systemic resistance responses,causing a lack of pathogenicity to plants.

Additionally, plants are protected from numerous classes of plantpathogen by responses that are similar to systemic acquired resistanceand rhizobacteria-induced systemic resistance. Trichoderma spp. caneffectively reduce diseases caused by some soil-borne plant pathogens.For example, the species T. harzianum, T. hamatum, and T. viride havefungicidal activity against Sclerotium, Rhizoctonia, Solani, Pythium,Fusarium, Cercospora, Ralstonia, Fragaria, Rhizopus, Botrytis,Colletotrichum, Magnaporthe, and many others. Moreover, some strains ofTrichoderma are able to effectively suppress the growth of some viraland bacterial plant and soil pathogens, as well as produce somesignificant nematocidal effects.

In addition to protecting plants from pathogens and pests, rootcolonization by Trichoderma spp. can enhance root growth anddevelopment, crop productivity, resistance to abiotic stresses, andbioavailability of nutrients.

In certain embodiments, the microorganisms are bacteria, includingGram-positive and Gram-negative bacteria. The bacteria may be, forexample Agrobacterium (e.g., A. radiobacter), Azotobacter (A.vinelandii, A. chroococcum), Azospirillum (e.g., A. brasiliensis),Bacillus (e.g., B. amyloliquefaciens, B. circulans, B. firmus, B.laterosporus, B. lichenmformis, B. megaterium, Bacillus mucilaginosus,B. subtilis), Frateuria (e.g., F. aurantia), Microbacterium (e.g., M.laevaniformans), myxobacteria (e.g., Myxococcus xanthus, Stignatellaaurantiaca, Sorangium cellulosum, Minicystis rosea), Pantoea (e.g., P.agglomerans), Pseudomonas (e.g., P. aeruginosa, P. chlororaphis subsp.aureofaciens (Kluyver), P. putida), Rhizobium spp., Rhodospirillum(e.g., R. rubrum), Sphingomonas (e.g., S. paucimobilis), and/orThiobacillus thiooxidans (Acidothiobacillus thiooxidans).

In a specific embodiment, the microorganism is Bacillusamyloliquefaciens, such as, for example, the strain B. amyloliquefacienssubsp. locus. In some embodiments, the Bacillus microbe can solubilizephosphorus compounds in the soil.

In one embodiment, the microorganism is a mycobacterium, orslime-forming bacteria. Specifically, in one embodiment, themycobacterium is a Myxococcus spp. bacterium, e.g., M. xanthus.

In certain embodiments, the microorganism is one that is capable offixing and/or solubilizing nitrogen, potassium, phosphorous and/or othermicronutrients in soil.

In one embodiment, the microorganism is a nitrogen-fixing microorganism,or a diazotroph, selected from species of, for example, Azospirillum,Azotobacter, Chlorobiaceae, Cyanothece, Frankia, Klebsiella, rhizobia,Trichodesmium, and some Archaea. In a specific embodiment, thenitrogen-fixing bacteria is Azotobacter vinelandii.

In another embodiment, the microorganism is a potassium-mobilizingmicroorganism, or KMB, selected from, for example, Bacillusmucilaginosus, Frateuria aurantia or Glomus mosseae. In a specificembodiment, the potassium-mobilizing microorganism is Frateuriaaurantia.

Additional microbes can include, for example, Pseudomonas chlororaphis,Wickerhamomyces anomalus, Starmerella bombicola, Saccharomycesboulardii, Pichia occidentalis, Pichia kudriavzevii, and/or Meyerozymaguilliermondii.

In one embodiment, the combination of microorganisms applied to a plantand/or its surrounding environment is customized for a given plantand/or environment. Advantageously, in some embodiments, the combinationof microbes work synergistically with one another to enhance planthealth, growth and/or yields.

Preparation of Microbe-Based Products

One microbe-based product of the subject invention is simply thefermentation medium containing the microorganisms and/or the microbialmetabolites produced by the microorganisms and/or any residualnutrients. The product of fermentation may be used directly withoutextraction or purification. If desired, extraction and purification canbe easily achieved using standard extraction and/or purification methodsor techniques described in the literature.

The microorganisms in the microbe-based products may be in an active orinactive form, or in the form of vegetative cells, reproductive spores,conidia, mycelia, hyphae, or any other form of microbial propagule. Themicrobe-based products may also contain a combination of any of theseforms of a microorganism.

In one embodiment, different strains of microbe are grown separately andthen mixed together to produce the microbe-based product. The microbescan, optionally, be blended with the medium in which they are grown anddried prior to mixing.

In one embodiment, the different strains are not mixed together, but areapplied to a plant and/or its environment as separate microbe-basedproducts.

The microbe-based products may be used without further stabilization,preservation, and storage. Advantageously, direct usage of thesemicrobe-based products preserves a high viability of the microorganisms,reduces the possibility of contamination from foreign agents andundesirable microorganisms, and maintains the activity of theby-products of microbial growth.

Upon harvesting the microbe-based composition from the growth vessels,further components can be added as the harvested product is placed intocontainers or otherwise transported for use. The additives can be, forexample, buffers, carriers, other microbe-based compositions produced atthe same or different facility, viscosity modifiers, preservatives,nutrients for microbe growth, surfactants, emulsifying agents,lubricants, solubility controlling agents, tracking agents, solvents,biocides, antibiotics, pH adjusting agents, chelators, stabilizers,ultra-violet light resistant agents, other microbes and other suitableadditives that are customarily used for such preparations.

In one embodiment, buffering agents including organic and amino acids ortheir salts, can be added. Suitable buffers include citrate, gluconate,tartarate, malate, acetate, lactate, oxalate, aspartate, malonate,glucoheptonate, pyruvate, galactarate, glucarate, tartronate, glutamate,glycine, lysine, glutamine, methionine, cysteine, arginine and a mixturethereof. Phosphoric and phosphorous acids or their salts may also beused. Synthetic buffers are suitable to be used but it is preferable touse natural buffers such as organic and amino acids or their saltslisted above.

In a further embodiment, pH adjusting agents include potassiumhydroxide, ammonium hydroxide, potassium carbonate or bicarbonate,hydrochloric acid, nitric acid, sulfuric acid or a mixture.

The pH of the microbe-based composition should be suitable for themicroorganism(s) of interest. In a preferred embodiment, the pH of thecomposition is about 3.5 to 7.0, about 4.0 to 6.5, or about 5.0.

In one embodiment, additional components such as an aqueous preparationof a salt, such as sodium bicarbonate or carbonate, sodium sulfate,sodium phosphate, sodium biphosphate, can be included in theformulation.

In certain embodiments, an adherent substance can be added to thecomposition to prolong the adherence of the product to plant parts.Polymers, such as charged polymers, or polysaccharide-based substancescan be used, for example, xanthan gum, guar gum, levan, xylinan, gellangum, curdlan, pullulan, dextran and others. In preferred embodiments,commercial grade xanthan gum is used as the adherent.

The concentration of the gum should be selected based on the content ofthe gum in the commercial product. If the xanthan gum is highly pure,then 0.001% (w/v—xanthan gum/solution) is sufficient.

In one embodiment, glucose, glycerol and/or glycerin can be added to themicrobe-based product to serve as, for example, an osmoticum duringstorage and transport. In one embodiment, molasses can be included.

In one embodiment, prebiotics can be added to and/or appliedconcurrently with the microbe-based product to enhance microbial growth.Suitable prebiotics, include, for example, kelp extract, fulvic acid,chitin, humate and/or humic acid. In a specific embodiment, the amountof prebiotics applied is about 0.1 L/acre to about 0.5 L/acre, or about0.2 L/acre to about 0.4 L/acre.

Optionally, the product can be stored prior to use. The storage time ispreferably short. Thus, the storage time may be less than 60 days, 45days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2days, 1 day, or 12 hours. In a preferred embodiment, if live cells arepresent in the product, the product is stored at a cool temperature suchas, for example, less than 20° C., 15° C., 10° C., or 5° C.

Local Production of Microbe-Based Products

In certain embodiments of the subject invention, a microbe growthfacility produces fresh, high-density microorganisms and/or microbialgrowth by-products of interest on a desired scale. The microbe growthfacility may be located at or near the site of application. The facilityproduces high-density microbe-based compositions in batch,quasi-continuous, or continuous cultivation.

The microbe growth facilities of the subject invention can be located atthe location where the microbe-based product will be used (e.g., acitrus grove). For example, the microbe growth facility may be less than300, 250, 200, 150, 100, 75, 50, 25, 15, 10, 5, 3, or 1 mile from thelocation of use.

Because the microbe-based product can be generated locally, withoutresort to the microorganism stabilization, preservation, storage andtransportation processes of conventional microbial production, a muchhigher density of microorganisms can be generated, thereby requiring asmaller volume of the microbe-based product for use in the on-siteapplication or which allows much higher density microbial applicationswhere necessary to achieve the desired efficacy. This allows for ascaled-down bioreactor (e.g., smaller fermentation vessel, smallersupplies of starter material, nutrients and pH control agents), whichmakes the system efficient and can eliminate the need to stabilize cellsor separate them from their culture medium. Local generation of themicrobe-based product also facilitates the inclusion of the growthmedium in the product. The medium can contain agents produced during thefermentation that are particularly well-suited for local use.

Locally-produced high density, robust cultures of microbes are moreeffective in the field than those that have remained in the supply chainfor some time. The microbe-based products of the subject invention areparticularly advantageous compared to traditional products wherein cellshave been separated from metabolites and nutrients present in thefermentation growth media. Reduced transportation times allow for theproduction and delivery of fresh batches of microbes and/or theirmetabolites at the time and volume as required by local demand.

The microbe growth facilities of the subject invention produce fresh,microbe-based compositions, comprising the microbes themselves,microbial metabolites, and/or other components of the medium in whichthe microbes are grown. If desired, the compositions can have a highdensity of vegetative cells or propagules, or a mixture of vegetativecells and propagules.

Advantageously, the compositions can be tailored for use at a specifiedlocation. In one embodiment, the microbe growth facility is located on,or near, a site where the microbe-based products will be used (e.g., acitrus grove).

Advantageously, these microbe growth facilities provide a solution tothe current problem of relying on far-flung industrial-sized producerswhose product quality suffers due to upstream processing delays, supplychain bottlenecks, improper storage, and other contingencies thatinhibit the timely delivery and application of, for example, a viable,high cell-count product and the associated medium and metabolites inwhich the cells are originally grown.

The microbe growth facilities provide manufacturing versatility by theirability to tailor the microbe-based products to improve synergies withdestination geographies. Advantageously, in preferred embodiments, thesystems of the subject invention harness the power ofnaturally-occurring local microorganisms and their metabolic by-productsto improve agricultural production.

The cultivation time for the individual vessels may be, for example,from 1 to 7 days or longer. The cultivation product can be harvested inany of a number of different ways.

Local production and delivery within, for example, 24 hours offermentation results in pure, high cell density compositions andsubstantially lower shipping costs. Given the prospects for rapidadvancement in the development of more effective and powerful microbialinoculants, consumers will benefit greatly from this ability to rapidlydeliver microbe-based products.

Methods of Enhancing Plant Root Health and Immune Health

In preferred embodiments, a method is provided for enhancing planthealth, growth and/or yields, wherein a combination of beneficialmicroorganisms, and/or their growth by-products, are applied to a plantand/or its surrounding environment. In some embodiments, multiple plantsand/or their surrounding environments are treated according to thesubject methods.

As used herein, a plant's “surrounding environment” means the soiland/or other medium in which the plant is growing, which can include therhizosphere. In certain embodiments, the surrounding environment doesnot extend past, for example, a radius of at least 5 miles, 1 mile,1,000 feet, 500 feet, 300 feet, 100 feet, 10 feet, 8 feet, or 6 feetfrom the plant.

In specific embodiments, the methods can comprise applying a firstmicroorganism and a second microorganism, and/or a growth by-product ofone or both of these microorganisms, to the plant and/or its surroundingenvironment. Preferably, the first microorganism is a Trichoderma spp.fungus and the second microorganism is a Bacillus spp. bacterium. Inspecific embodiments, the method comprises applying a soil treatmentcomposition according to the subject description to the plant and/or itsenvironment.

In one embodiment, the method comprises cultivating the first and secondmicroorganisms separately and then combining them to produce one soiltreatment composition. In one embodiment, the first and secondmicroorganisms are not blended together into one product, but areapplied to the plant and/or its environment as separate treatments.

To improve or stabilize the effects of the treatment composition, it canbe blended with suitable adjuvants and then used as such or afterdilution if necessary. In preferred embodiments, the composition isformulated as a dry powder or as granules, which can be mixed with waterand other components to form a liquid product.

In one embodiment, additional microorganisms can be appliedcontemporaneously with the Trichoderma and/or Bacillus. For example, amycobacterium such as Myxococcus xanthus can also be applied, and/or oneor more microorganisms capable of fixing, mobilizing and/or solubilizingnitrogen, potassium, phosphorous (or phosphate) and/or othermicronutrients in soil. In one embodiment, a nitrogen-fixing microbe,such as, for example, Azotobacter vinelandii, can also be applied. Inanother embodiment, a potassium-mobilizing microbe, such as, forexample, Frateuria aurantia can also be applied.

In some embodiments, the methods further comprise applying materialswith the composition to enhance microbe growth during application (e.g.,nutrients and/or prebiotics to promote microbial growth). In oneembodiment, nutrient sources can include, for example, sources ofnitrogen, potassium, phosphorus, magnesium, proteins, vitamins and/orcarbon. In one embodiments, prebiotics can include, for example, kelpextract, fulvic acid, chitin, humate and/or humic acid.

In one embodiment, the method can enhance plant health, growth and/oryields by enhancing root health and growth. More specifically, in oneembodiment, the methods can be used to improve the properties of therhizosphere in which a plant's roots are growing, for example, thenutrient and/or moisture retention properties.

Additionally, in one embodiment, the method can be used to inoculate arhizosphere with one or more beneficial microorganisms. For example, inpreferred embodiments, the microbes of the soil treatment compositioncan colonize the rhizosphere and provide multiple benefits to the plantwhose roots are growing therein, including protection and nourishment.Advantageously, in one embodiment, the subject methods can be used toenhance health, growth and/or yields in plants having compromised immunehealth due to an infection from a pathogenic agent or from anenvironmental stressor, such as, for example, drought. Thus, in certainembodiments, the subject methods can also be used for improving theimmune health, or immune response, of plants.

As used herein, “applying” a composition or product refers to contactinga composition or product with a target or site such that the compositionor product can have an effect on that target or site. The effect can bedue to, for example, microbial growth and/or interaction with a plant,as well as the action of a metabolite, enzyme, biosurfactant or othermicrobial growth by-product. Applying can also include “treating” atarget or site with a composition.

Application can further include contacting the microbe-based productdirectly with a plant, plant part, and/or the plant's surroundingenvironment (e.g., the soil or the rhizosphere). The microbe-product canbe applied as a seed treatment or to the soil surface, or to the surfaceof a plant or plant part (e.g., to the surface of the roots, tubers,stems, flowers, leaves, fruit, or flowers). It can be sprayed, poured,sprinkled, injected or spread as liquid, dry powder, dust, granules,microgranules, pellets, wettable powder, flowable powder, emulsions,microcapsules, oils, gels, pastes or aerosols.

In a specific embodiment, the composition is contacted with one or moreroots of the plant. The composition can be applied directly to theroots, e.g., by spraying or dunking the roots, and/or indirectly, e.g.,by administering the composition to the soil in which the plant grows(e.g., the rhizosphere). The composition can be applied to the seeds ofthe plant prior to or at the time of planting, or to any other part ofthe plant and/or its surrounding environment.

In certain embodiments, the compositions provided herein are applied tothe soil surface without mechanical incorporation. The beneficial effectof the soil application can be activated by rainfall, sprinkler, flood,or drip irrigation, and subsequently delivered to, for example, theroots of plants.

Plants and/or their environments can be treated at any point during theprocess of cultivating the plant. For example, the soil treatmentcomposition can be applied to the soil prior to, concurrently with, orafter the time when seeds are planted therein. It can also be applied atany point thereafter during the development and growth of the plant,including when the plant is flowering, fruiting, and during and/or afterabscission of leaves.

In one embodiment, the method can be used in a large scale agriculturalsetting. The method can comprise administering the soil treatmentcomposition into a tank connected to an irrigation system used forsupplying water, fertilizers or other liquid compositions to a crop,orchard or field. Thus, the plant and/or soil surrounding the plant canbe treated with the soil treatment composition via, for example, soilinjection, soil drenching, or using a center pivot irrigation system, orwith a spray over the seed furrow, or with sprinklers or dripirrigators. Advantageously, the method is suitable for treating hundredsof acres of crops, orchards or fields at one time.

In one embodiment, the method can be used in a smaller scale setting,such as in a home garden or greenhouse. In such cases, the method cancomprise spraying a plant and/or its surrounding environment with thesoil treatment composition using a handheld lawn and garden sprayer. Thecomposition can be mixed with water, and optionally, other lawn andgarden treatments, such as fertilizers and pesticides. The compositioncan also be mixed in a standard handheld watering can and poured ontosoil.

In certain embodiments, the plant receiving treatment is healthy.Advantageously, the subject invention can be useful in enhancing theimmune response of a plant having a compromised immune system, forexample, because the plant is affected by disease and/or diseasesymptoms.

For example, the plant may be affected by a pathogenic strain ofPseudomonas (e.g., P. savastanoi, P. syringae pathovars); Ralstoniasolanacearum; Agrobacterium (e.g., A. tumefaciens); Xanthomonas (e.g.,X. oryzae pv. Oryzae, X. campestris pathovars, X. axonopodis pathovars);Erwinia (e.g., E. amylovora); Xylella (e.g., X. fastidiosa); Dickeya(e.g., D. dadantdi and D. solani); Pectobacterium (e.g., P. carotovorumand P. atrosepticum); Clavibacter (e.g., C. michiganensis and C.sepedonicus); Candidatus Liberibacter asiaticus; Pantoea; Burkholderia;Acidovorax; Streptomyces; Spiroplasma; and/or Phytoplasma; as well ashuanglongbing (HLB, citrus greening disease), citrus canker disease,citrus bacterial spot disease, citrus variegated chlorosis, brown rot,citrus root rot, citrus and black spot disease.

In one embodiment, the methods are used to enhance the health, growthand/or yields of citrus trees affected by citrus greening disease and/orcitrus canker disease.

The present invention can be used to enhance health, growth and/oryields of plants and/or crops in, for example, agriculture,horticulture, greenhouses, landscaping, and the like.

In one embodiment, the subject invention can also be used for improvingone or more qualities of soil, thereby enhancing the performance of thesoils for agricultural, home and gardening purposes.

In certain embodiments, the soil treatment composition may also beapplied so as to promote colonization of the roots and/or rhizosphere aswell as the vascular system of the plant in order to enhance planthealth and vitality. Thus, nutrient-fixing microbes such as Rhizobiumand/or Mycorrhizae can be promoted, as well as other beneficialendogenous and exogenous microbes, and/or their by-products that promotecrop growth, health and/or yield.

In one embodiment, the method can be used for enhancing penetration ofbeneficial molecules through the outer layers of root cells.

The subject invention can be used to improve any number of qualities inany type of soil, for example, clay, sandy, silty, peaty, chalky, loamsoil, and/or combinations thereof. Furthermore, the methods andcompositions can be used for improving the quality of dry, waterlogged,porous, depleted, compacted soils and/or combinations thereof.

In one embodiment, the method can be used for improving the drainageand/or dispersal of water in waterlogged soils. In one embodiment, themethod can be used for improving water retention in dry soil.

In one embodiment, the method can be used for improving nutrientretention in porous and/or depleted soils.

In one embodiment, the method controls pathogenic bacteria themselves.In one embodiment, the method works by enhancing the immune health ofplants to increase the ability to fight off infections.

In yet another embodiment, the method controls pests that might act asvectors or carriers for pathogenic bacteria, such as flies, aphids,ants, beetles, and whiteflies. Thus, the subject methods can prevent thespread of plant pathogenic bacteria by controlling, e.g., killing, thesecarrier pests.

The microbe-based products can be used either alone or in combinationwith other compounds for efficient enhancement of plant health, growthand/or yields, as well as other compounds for efficient treatment andprevention of plant pathogenic pests. For example, the methods can beused concurrently with sources of nutrients and/or micronutrients forenhancing plant and/or microbe growth, such as magnesium, phosphate,nitrogen, potassium, selenium, calcium, sulfur, iron, copper, and zinc;and/or one or more prebiotics, such as kelp extract, fulvic acid,chitin, humate and/or humic acid. The exact materials and the quantitiesthereof can be determined by a grower or an agricultural scientisthaving the benefit of the subject disclosure.

The compositions can also be used in combination with other agriculturalcompounds and/or crop management systems. In one embodiment, thecomposition can optionally comprise, and/or be applied with, forexample, natural and/or chemical pesticides, repellants, herbicides,fertilizers, water treatments, non-ionic surfactants and/or soilamendments.

In one embodiment, the subject compositions are compatible for use withagricultural compounds characterized as antiscalants, such as, e.g.,hydroxyethylidene diphosphonic acid;

bactericides, such as, e.g., streptomycin sulfate and/or Galltrol® (A.radiobacter strain K84);

biocides, such as, e.g., chlorine dioxide, didecyldimethyl ammoniumchloride, halogenated heterocyclic, and/or hydrogen dioxide/peroxyaceticacid;

fertilizers, such as, e.g., N-P-K fertilizers, calcium ammonium nitrate17-0-0, potassium thiosulfate, nitrogen (e.g., 10-34-0, Kugler KQ-XRN,Kugler KS-178C, Kugler KS-2075, Kugler LS 6-24-6S, UN 28, UN 32), and/orpotassium;

fungicides, such as, e.g., chlorothalonil, manicozebhexamethylenetetramine, aluminum tris, azoxystrobin, Bacillus spp.(e.g., B. licheniformis strain 3086, B. subtilis, B. subtilis strain QST713), benomyl, boscalid, pyraclostrobin, captan, carboxin, chloroneb,chlorothalonil, copper culfate, cyazofamid, dicloran, dimethomorph,etridiazole, thiophanate-methyl, fenamidone, fenarimol, fludioxonil,fluopicolide, flutolanil, iprodione, mancozeb, maneb, mefanoxam,fludioxonil, mefenoxam, metalaxyl, myclobutanil, oxathiapiprolin,pentachloronitrobenzene (quintozene), phosphorus acid, propamocarb,propanil, pyraclostrobin, Reynoutria sachalinensis, Streptomyces spp.(e.g., S. griseoviridis strain K61, S. lydicus WYEC 108), sulfur, urea,thiabendazole, thiophanate methyl, thiram, triadimefon, triadimenol,and/or vinclozolin;

growth regulators, such as, e.g., ancymidol, chlormequat chloride,diaminozide, paclobutrazol, and/or uniconazole;

herbicides, such as, e.g., glyphosate, oxyfluorfen, and/orpendimethalin;

insecticides, such as, e.g., acephate, azadirachtin, B. thuringiensis(e.g., subsp. israelensis strain AM 65-52), Beauveria bassiana (e.g.,strain GHA), carbaryl, chlorpyrifos, cyantraniliprole, cyromazine,dicofol, diazinon, dinotefuran, imidacloprid, Isaria fumosorosae (e.g.,Apopka strain 97), lindane, and/or malathion;

water treatments, such as, e.g., hydrogen peroxide (30-35%), phosphonicacid (5-20%), and/or sodium chlorite;

as well as glycolipids, lipopeptides, deet, diatomaceous earth,citronella, essential oils, mineral oils, garlic extract, chili extract,and/or any known commercial and/or homemade pesticide that is determinedto be compatible by the skilled artisan having the benefit of thesubject disclosure.

Preferably, the composition does not comprise and/or is not appliedsimultaneously with, or within 7 to 10 days before or after, applicationof the following compounds: benomyl, dodecyl dimethyl ammonium chloride,hydrogen dioxide/peroxyacetic acid, imazilil, propiconazole,tebuconazole, or triflumizole.

In certain embodiments, the compositions and methods can be used toenhance the effectiveness of other compounds, for example, by enhancingthe penetration of a pesticidal compound into a plant or pest, orenhancing the bioavailability of a nutrient to plant roots. Themicrobe-based products can also be used to supplement other treatments,for example, antibiotic treatments. Advantageously, the subjectinvention helps reduce the amount of antibiotics that must beadministered to a crop or plant in order to be effective at treatingand/or preventing bacterial infection.

In one embodiment, the methods and compositions according to the subjectinvention lead to an increase in one or more of: root mass, trunkdiameter, canopy density, brix value, chlorophyll content, flower countand/or leaf tissue nitrogen levels of a plant, by about 5%, 10%, 20%,30%, 40%, 50%, 60% 70%, 80%, 90%, 100%, 150%, 200%, or more, compared toa plant growing in an untreated environment.

In certain embodiments, the methods and compositions according to thesubject invention lead to an increase in crop yield by about 5%, 10%,20%, 30%, 40%, 50%, 60% 70%, 80%, 90%, 100%, 150%, 200%, or more,compared to untreated crops.

In one embodiment, the methods and compositions according to the subjectinvention lead to a reduction in the number of pests on a plant or in aplant's surrounding environment by about 55%, 10%, 20%, 30%, 40%, 50%,60% 70%, 80%, 90%, 100%, 150%, 200%, or more, compared to a plantgrowing in an untreated environment.

In one embodiment, the methods and compositions according to the subjectinvention reduce damage to a plant caused by pests by about 5%, 10%,20%, 30%, 40%, 50%, 60% 70%, 80%, 90%, 100%, 150%, 200%, or more,compared to plants growing in an untreated environment.

Target Plants

As used here, the term “plant” includes, but is not limited to, anyspecies of woody, ornamental or decorative, crop or cereal, fruit plantor vegetable plant, flower or tree, macroalga or microalga,phytoplankton and photosynthetic algae (e.g., green algae Chlamydomonasreinhardtii). “Plant” also includes a unicellular plant (e.g.,microalga) and a plurality of plant cells that are largelydifferentiated into a colony (e.g., volvox) or a structure that ispresent at any stage of a plant's development. Such structures include,but are not limited to, a fruit, a seed, a shoot, a stem, a leaf, aroot, a flower petal, etc. Plants can be standing alone, for example, ina garden, or can be one of many plants, for example, as part of anorchard, crop or pasture.

As used herein, “crop plants” refer to any species of plant or algaedible by humans or used as a feed for animals or fish or marineanimals, or consumed by humans, or used by humans (e.g., textile orcosmetics production), or viewed by humans (e.g., flowers or shrubs inlandscaping or gardens) or any plant or alga, or a part thereof, used inindustry or commerce or education.

Types of crop plants that can benefit from application of the productsand methods of the subject invention include, but are not limited to:row crops (e.g., corn, soy, sorghum, peanuts, potatoes, etc.), fieldcrops (e.g., alfalfa, wheat, grains, etc.), tree crops (e.g., walnuts,almonds, pecans, hazelnuts, pistachios, etc.), citrus crops (e.g.,orange, lemon, grapefruit, etc.), fruit crops (e.g., apples, pears,strawberries, blueberries, blackberries, etc.), turf crops (e.g., sod),ornamentals crops (e.g., flowers, vines, etc.), vegetables (e.g.,tomatoes, carrots, etc.), vine crops (e.g., grapes, etc.), forestry(e.g., pine, spruce, eucalyptus, poplar, etc.), managed pastures (anymix of plants used to support grazing animals).

Additional examples of plants for which the subject invention is usefulinclude, but are not limited to, cereals and grasses (e.g., wheat,barley, rye, oats, rice, maize, sorghum, corn), beets (e.g., sugar orfodder beets); fruit (e.g., grapes, strawberries, raspberries,blackberries, pomaceous fruit, stone fruit, soft fruit, apples, pears,plums, peaches, almonds, cherries or berries); leguminous crops (e.g.,beans, lentils, peas or soya); oil crops (e.g., oilseed rape, mustard,poppies, olives, sunflowers, coconut, castor, cocoa or ground nuts);cucurbits (e.g., pumpkins, cucumbers, squash or melons); fiber plants(e.g., cotton, flax, hemp or jute); citrus fruit (e.g., oranges, lemons,grapefruit or tangerines); vegetables (e.g., spinach, lettuce,asparagus, cabbages, carrots, onions, tomatoes, potatoes or bellpeppers); Lauraceae (e.g., avocado, Cinnamonium or camphor); and alsotobacco, nuts, herbs, spices, medicinal plants, coffee, eggplants,sugarcane, tea, pepper, grapevines, hops, the plantain family, latexplants, cut flowers and ornamentals.

In certain embodiments, the crop plant is a citrus plant. Examples ofcitrus plants according to the subject invention include, but are notlimited to, orange trees, lemon trees, lime trees and grapefruit trees.Other examples include Citrus maxima (Pomelo), Citrus medica (Citron),Citrus micrantha (Papeda), Citrus reticulata (Mandarin orange), Citrusparadisi (grapefruit), Citrus japonica (kumquat), Citrus australasica(Australian Finger Lime), Citrus australis (Australian Round lime),Citrus glauca (Australian Desert Lime), Citrus garrawayae (Mount WhiteLime), Citrus gracilis (Kakadu Lime or Humpty Doo Lime), Citrus inodora(Russel River Lime), Citrus warburgiana (New Guinea Wild Lime), Citruswintersii (Brown River Finger Lime), Citrus halimii (limau kadangsa,limau kedut kera), Citrus indica (Indian wild orange), Citrusmacroptera, and Citrus latipes, Citrus x aurantiifolia (Key lime),Citrus x aurantium (Bitter orange), Citrus x latifolia (Persian lime),Citrus x limon (Lemon), Citrus x limonia (Rangpur), Citrus x sinensis(Sweet orange), Citrus x tangerina (Tangerine), Imperial lemon, tangelo,orangelo, tangor, kinnow, kiyomi, Minneola tangelo, oroblanco, ugli,Buddha's hand, citron, bergamot orange, blood orange, calamondin,clementine, Meyer lemon, and yuzu.

In some embodiments, the crop plant is a relative of a citrus plant,such as orange jasmine, limeberry, and trifoliate orange (Citrustrifolata).

Additional examples of target plants include all plants that belong tothe superfamily Viridiplantae, in particular monocotyledonous anddicotyledonous plants including fodder or forage legumes, ornamentalplants, food crops, trees or shrubs selected from Acer spp., Actinidiaspp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostisstolonmfera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananascomosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp.,Asparagus officinalis, Avena spp. (e.g., A. sativa, A. fatua, A.byzantina, A. fatua var. sativa, A. hybrida), Averrhoa carambola,Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris,Brassica spp. (e.g., B. napus, B. rapa ssp. [canola, oilseed rape,turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica,Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissamacrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceibapentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrusspp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorussp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus,Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodiumspp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloaspp., Elaeis (e.g., E. guineensis, E. oleifera), Eleusine coracana,Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp.,Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficuscarica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp.(e.g., G. max, Soja hispida or Soja max), Gossypium hirsutum, Helianthusspp. (e.g., H. annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp.(e.g., H. vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa,Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis,Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica,Lycopersicon spp. (e.g., L. esculentum, L. lycopersicum, L. pyriforme),Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana,Mangifera indica, Manihot spp., Manilkara zapota, Medicago saliva,Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morusnigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopusspp., Oryza spp. (e.g., O. saliva, O. latifolia), Panicum miliaceum,Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp.,Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp.,Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp.,Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopisspp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis,Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinuscommunis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secalecereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g., S. tuberosum, S.integrifolium or S. lycopersicum), Sorghum bicolor, Spinacia spp.,Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao,Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticumspp. (e.g., T. aestivum, T. durum, T. turgidum, T. hybernum, T. macha,T. sativum, T. monococcum or T. vulgare), Tropaeolum minus, Tropaeolummajus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitisspp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.

Target plants can also include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosaliva), rice (Oryza saliva), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Target vegetable plants include tomatoes (Lycopersicon esculentum),lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), limabeans (Phaseolus limensis), peas (Lathyrus spp.), and members of thegenus Cucumis such as cucumber (C. sativus), cantaloupe (C.cantalupensis), and musk melon (C. melo). Ornamentals include azalea(Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus(Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.),daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum. Conifers that may be employed in practicing theembodiments include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata);Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis);Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firssuch as silver fir (Abies amabilis) and balsam fir (Abies balsamea); andcedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Plants of the embodiments include cropplants (for example, corn, alfalfa, sunflower, Brassica, soybean,cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), suchas corn and soybean plants.

Target turfgrasses include, but are not limited to: annual bluegrass(Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewings fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerate); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovine); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pretense); velvet bentgrass (Agrostis canine); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithii); Bermuda grass (Cynodon spp.); St. Augustine grass(Stenotaphrum secundalum); zoysia grass (Zoysia spp.); Bahia grass(Paspalum notatum); carpet grass (Axonopus affinis); centipede grass(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);seashore paspalum (Paspalum vaginatum); blue gramma (Boutelouagracilis); buffalo grass (Buchloe dactyloids); sideoats gramma(Bouteloua curtipendula).

Further plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,millet, etc. Oil-seed plants include cotton, soybean, safflower,sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, oliveetc. Leguminous plants include beans and peas. Beans include guar,locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, limabean, fava bean, lentils, chickpea, etc.

Further plants of interest include Cannabis (e.g., sativa, indica, andruderalis) and industrial hemp.

All plants and plant parts can be treated in accordance with theinvention. In this context, plants are understood as meaning all plantsand plant populations such as desired and undesired wild plants or cropplants (including naturally occurring crop plants). Crop plants can beplants that can be obtained by traditional breeding and optimizationmethods or by biotechnological and recombinant methods, or combinationsof these methods, including the transgenic plants and the plantvarieties.

Plant parts are understood as meaning all aerial and subterranean partsand organs of the plants such as shoot, leaf, flower and root, exampleswhich may be mentioned being leaves, needles, stalks, stems, flowers,fruit bodies, fruits and seeds, but also roots, tubers and rhizomes. Theplant parts also include crop material and vegetative and generativepropagation material, for example cuttings, tubers, rhizomes, slips andseeds.

In some embodiments, the plant is a plant infected by a pathogenicdisease or pest. In specific embodiments, the plant is infected withcitrus greening disease and/or citrus canker disease, and/or a pest thatcarries such diseases.

Examples

A greater understanding of the present invention and of its manyadvantages may be had from the following examples, given by way ofillustration. The following examples are illustrative of some of themethods, applications, embodiments and variants of the presentinvention. They are not to be considered as limiting the invention.Numerous changes and modifications can be made with respect to theinvention.

Example 1—Solid State Fermentation of Bacillus Microbes

For Bacillus spp. spore production, a wheat bran-based media is used.The media is spread onto stainless steel pans in a layer about 1 to 2inches think and sterilized.

Following sterilization, the pans are inoculated with seed culture.Optionally, added nutrients can be included to enhance microbial growth,including, for example, salts and/or carbon sources such as molasses,starches, glucose and sucrose. To increase the speed of growth andincrease the motility and distribution of the bacteria throughout theculture medium, potato extract or banana peel extract can be added tothe culture.

Spores of the Bacillus strain of choice are then sprayed or pipettedonto the surface of the substrate and the trays are incubated between32-40° C. Ambient air is pumped through the oven to stabilize thetemperature. Incubation for 48-72 hours can produce 1×10¹⁰ spores/gramor more of the strain.

Example 2—Solid State Fermentation of Fungal Spores

For growing Trichoderma spp., 250 g of nixtamilized corn flour is mixedwith deionized water and sterilized in a stainless steel pan, sealedwith a lid and pan bands. The corn flour medium is asepticallyinoculated with Trichoderma seed culture by spraying or pipetting. Thepans are then incubated at 30° C. for 10 days. After 10 days,approximately 10⁹ propagules/gram or more of Trichoderma can beharvested. Trichoderma propagules (conidia and/or hyphae) harvested fromone batch can treat, for example, 1,000 to 2,000 acres of land.

Example 3—Preparation of Microbe-Based Product

The microbes, substrate, and any residual nutrients that result fromproduction using the methods described in Examples 1 and 2 can beblended and/or micronized and dried to form granules or a powdersubstance. Different strains of microbe are produced separately and thenmixed together either before or after drying. A sealable pouch can beused to store and transport a product containing a mixture of 10⁹cells/g of T. harzianum and 10¹⁰ cells/g of B. amyloliquefaciens.Micronutrients, or other microbes similarly produced, can be added tothe product.

To prepare for use, the dry product is dissolved in water. Theconcentration can reach at least 5×10⁹ to 5×10¹⁰ cells/ml. The productis then diluted with water in a mixing tank to a concentration of 1×10⁶to 1×10⁷ cells/ml.

One bag can be used to treat approximately 20 acres of crop, or 10 acresof citrus grove.

Example 4—Orange Tree Root Trial, Ft. Basinger Florida

A soil treatment composition of the subject invention (referred to asRhizolizer™), comprising 37.5 ml Trichoderma harzianum culture and 37.5ml of Bacillus amyloliquefaciens subsp. locus culture with 2 quarts ofkelp extract, was applied in two repeats to orange plants in a Ft.Basinger, Fla. grove. Wet (fresh) root mass and dry root mass werecompared with those of untreated control plants.

As shown in Table 1, average root flush measurements of plants treatedwith Rhizolizer™ were higher for both dry weight (FIG. 1A) and freshweight than those grown using standard growers practice (untreatedcontrol).

TABLE 1 Average root flush measurements of untreated control (UTC) andRhizolizer ™ (Treatment B). Treatment Rep Dry weight Fresh weight CodeTreatment No (g) (g) UTC UTC 1 8.75 33.97 UTC UTC 2 12.26 45.77 Sum21.01 79.74 TREATMENT TDH10/ 1 19.8 80.18 B BCA10 TREATMENT TDH10/ 216.79 67.64 B BCA10 Sum 36.59 147.82

Example 5—Grapefruit Tree Root Trial, Ft. Pierce Florida

A soil treatment composition of the subject invention (referred to asRhizolizer™), comprising 37.5 ml Trichoderma harzianum culture and 37.5ml of Bacillus amyloliquefaciens subsp. locus culture with 2 quarts ofkelp extract, was applied in four repeats to grapefruit plants in Ft.Pierce, Fla. groves. Repeats occurred every two months. After the fourthrepeat, wet (fresh) root mass and dry root mass were compared with thoseof untreated control plants.

Average root flush measurements of plants treated with Rhizolizer™ werehigher for both dry weight (FIG. 1B) and fresh/wet weight than thosegrown using standard growers practice (untreated control).

Example 6—Florida Citrus Trials

Citrus plants in various counties of Florida were treated with a soiltreatment composition according to an embodiment of the subjectinvention and compared with untreated control plants (grower'spractice). Root mass/weight, shoot growth, canopy density, trunkcaliper, fruit size, fruit Brix rating, and yield (weight boxes) werestudied.

FIG. 2 shows the dry root mass of grapefruit plants in St. Lucie County.The soil treatment composition was applied quarterly, for a total of 8treatments (1.5 fl. oz/acre each). A 153% increase in dry root mass wasobserved compared with grower's practice plants.

FIG. 3A shows the dry root mass (g/sample) of Hamlin orange trees inHighlands County. The soil treatment composition was applied quarterly,for a total of 3 treatments (1.5 fl. oz./acre each). A 76% increase indry root mass was observed compared with grower's practice plants. FIG.3B shows the dry root mass (g/sample) of white grapefruit trees in St.Lucie County. The soil treatment composition was applied quarterly, fora total of 3 treatments (1.5 fl. oz./acre each). An 82% increase in dryroot mass was observed compared with grower's practice plants.

FIG. 4A shows average root weight (g) of Hamlin orange trees in PolkCounty. The soil treatment composition was applied quarterly, for atotal of 4 treatments (1.5 fl. oz./acre for the first three treatments;3 fl. oz./acre for the final treatment). A 93% increase in average rootweight was observed. FIG. 4B shows average root weight (g) of Valenciaorange trees in Collier County. The soil treatment composition wasapplied quarterly, for a total of 4 treatments (1.5 fl. oz./acre for thefirst three treatments; 3 fl. oz./acre for the final treatment). A 35%increase in average root weight was observed.

FIGS. 5A-5B show percent of trees with new shoot growth (5A) and averageshoot count (5B) in young Valencia orange trees in Highlands County. Thesoil treatment composition was applied bi-monthly, for a total of 6treatments (1.5 fl. oz./acre each). A 73% increase in percent trees withnew shoot growth was observed, and a total of 38 more shoots on averagewere observed for young trees.

FIGS. 6A-6B show increase in canopy density for mature Hamlin orangetrees (6A) and young Hamlin orange trees (6B) in Highlands County. Thesoil treatment composition was applied quarterly, for a total of 3treatments (1.5 fl. oz./acre each). A 19% increase in visual canopyrating was observed for mature trees, and a 33% increase was observedfor young trees.

FIG. 7 shows the average caliper change in young Valencia orange treesin Highlands County. The soil treatment composition was appliedbi-monthly, for a total of 4 treatments (1.5 fl. oz./acre each). A 102%increase in caliper was observed for treated plants between February andOctober, while only an 81% increase in caliper was observed for grower'spractice plants.

FIGS. 8A-8B show fruit weight for white grapefruit trees (8A) anddiameter for ruby red grapefruit trees (8B) in Charlotte County. Thesoil treatment composition was applied quarterly, for a total of 4treatments (1.5 fl. oz./acre each). A 6% increase in weight was observedfor the white grapefruits, and a 2% increase in fruit diameter wasobserved for the ruby red.

FIGS. 9A-9B show Brix ratings for white grapefruits in Lucie County (9A)and Charlotte County (9B). The soil treatment composition was appliedbi-monthly in Lucie County, for a total of 5 treatments (1.5 fl. oziacreeach), and quarterly in Charlotte county, for a total of threetreatments (1.5 fl. oz./acre each). A 4% increase in degrees Brix wasobserved for the Lucie County grapefruits, and an 8% increase wasobserved for the Charlotte county grapefruits.

FIGS. 10A-10B show weight boxes for Hamlin oranges grown by twodifferent growers in Lake County. The soil treatment composition wasapplied quarterly for a total of 4 treatments (1.5 fl. oz./acre each). A42% increase was observed by Grower 1 (10A) and a 14% increase wasobserved by Grower 2 (10B).

FIGS. 11A-11B show total harvest yields in pounds solids for Hamlinoranges grown by two different growers in Lake County. The soiltreatment composition was applied quarterly for a total of 4 treatments(1.5 fl. oz./acre each). A 32% increase was observed by Grower 1 (11A)and a 9% increase was observed by Grower 2 (11B).

FIGS. 12A-12B show total harvest yields in pounds solids per box (12A)and per acre (12B) for Hamlin oranges in Polk County. The soil treatmentcomposition was applied bi-monthly for a total of 4 treatments (1.5 fl.oz./acre each). A 5% increase in pounds solid/box was observed, and a 6%increase in pounds solid/acre was observed.

Example 7—Tomato Field Trial, California Central Valley

A soil treatment composition of the subject invention (referred to asRhizolizer™), comprising 37.5 ml Trichoderma harzianum culture and 37.5ml of Bacillus amyloliquefaciens subsp. locus culture with 2 quarts ofkelp extract, was applied in three repeats, ranging from 26 to 33 daysapart, to tomato plants. About 29-32 days after the third repeatapplication, the tomato fruits were harvested and yields were measured.Harvest yields in tons per acre were measured for 10 ft×5 ft (0.001acre) plots.

As shown in FIG. 13A, red fruit yields (bars) and total fruit yields(diamonds) were compared between plants treated with the treatmentsshown in Table 2.

TABLE 2 Soil treatments for tomato plant study. 1 Untreated control 237.85 ml Trichoderma harzianum culture + 37.85 ml of Bacillusamyloliquefaciens subsp. locus culture 3 37.85 ml Trichoderma harzianumculture + 37.85 ml of Bacillus amyloliquefaciens subsp. locus culture +1.89 ml of potato extract 4 37.85 ml Trichoderma harzianum culture +37.85 ml of Bacillus amyloliquefaciens subsp. locus culture + 1 L ofhumate + 2 lb. of molasses 5 Rhizolizer ™ 6 2 pt. of Terra Treat ™ 7Commercial check (Viusid at 3, 1.5, and 2.3 fl oz.).

As shown in FIGS. 13A-13B, Rhizolizer™ treated tomato plants produced 42tons of red fruit per acre (out of 48 total tons of fruit per acre),while the untreated control produced 30 tons of red fruit per acre (outof 38 total tons of fruit per acre).

Example 8—Tomato Field Trial, Fresno County, California

Tomato plants in Fresno County, Calif., were treated with a soiltreatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”) and compared with untreated control (standardgrower's practice) plants. Yield, Brix rating, and dry root mass weremeasured.

FIG. 14A shows total marketable fruit yields in mT per acre for tomatoplants treated monthly with the soil treatment composition, for a totalof 3 treatments (3 fl. oz./acre each).

A 40% increase in yield was observed compared with grower's practiceplants.

FIG. 14B shows Brix rating for tomato plants treated monthly with thesoil treatment composition, for a total of 3 treatments (3 fl. oz./acreeach). A 26% increase in degrees Brix was observed over untreatedcontrol plants.

FIG. 15A shows dry root mass (g) of tomato plants treated once with thesoil treatment composition at 3 fl. oz./acre. A 50% increase in rootmass was observed over untreated control plants. FIG. 15B shows tomatoroots harvested from a tomato plant. The roots from the treated plant(bottom image) were visibly denser than the grower's practice plant (topimage).

Example 9—Trunk Growth Study in California Almond Trees

Almond trees in Ceres, Calif. were measured for trunk diameter growthwhen treated with eight different soil treatments over the course of 5months. The eight soil treatments were as shown in Table 3.

TABLE 3 Soil treatments for almond tree trunk growth measurement. 1Positive control 2 Trichoderma harzianum culture 0.001% v/v 3Trichoderma harzianum culture 0.01% v/v 4 Trichoderma harzianum culture0.1% v/v 5 Trichoderma harzianum culture 0.001% v/v + Bacillusamyloliquefaciens subsp. locus culture 0.001% v/v 6 Trichodermaharzianum culture 0.01% v/v + Bacillus amyloliquefaciens subsp. locusculture 0.01% v/v (Rhizolizer ™) 7 Trichoderma harzianum culture 0.1%v/v + Bacillus amyloliquefaciens subsp. locus culture 0.1% v/v 8Untreated control

FIG. 16A shows the results of tree trunk diameter measurements, wheretreatment #6 from the left (Rhizolizer™) showed a change in trunkdiameter of 1.25 mm, while untreated control only measured a 0.96 mmincrease in trunk diameter. FIG. 16B shows the trunk growth over time oftrees treated with the Rhizolizer™ treatment.

Example 10—Almond Bloom/Nut Set Trials

Almond trees in San Joaquin County, Calif. were treated with a soiltreatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”), and compared with untreated control (standardgrower's practice) plants. The soil treatment composition was appliedtwice, post-harvest, at 3 fl. oz./acre each.

After blooming, the number of set nuts was measured. A 21% increase inset nuts was observed for the treated trees over the untreated controltrees (FIG. 17).

Example 11—Watermelon Field Trials

Watermelon plants in Hillsborough County, Fla. were treated twice withRhizolizer™ (one treatment per month for two months) and compared withstandard grower's practice watermelon plants. FIGS. 18A-18F show theroot size (A-B), flower count (C), fruit count (D), yields (E), weightand Brix rating (F) for treated plants compared with untreated grower'spractice plants, where increases were observed for each data set.

Example 12—Cantaloupe Field Trials

Cantaloupe plants in Polk County, Fla. were treated twice withRhizolizer™ (one treatment per month for two months) and compared withstandard grower's practice. FIG. 19A shows the increase in flower countand FIG. 19B shows the average harvestable yield for treated plantscompared with untreated grower's practice plants, where increases wereobserved for each data set.

Example 13—Potato Field Trials

Potato plants in various locations were treated with a soil treatmentcomposition according to an embodiment of the subject invention(“Rhizolizer™”) and compared with untreated control (standard grower'spractice) plants. Emergence after planting, yields, and quality ofpotatoes were measured.

FIGS. 20A-20B show potato plant emergence in Imperial County, Calif. foryellow rose russet potatoes planted 5 weeks after planting (A) andmultiple cultivars 6 weeks after planting (B). The soil treatmentcomposition was applied once at the time of planting, at 1 fl. oz./acre.At 5 weeks, the yellow rose russet showed a 246% increase over grower'spractice plants (plants/linear 867 ft.). At 6 weeks, the multi-cultivarsshowed a 56% increase (plants/linear 1250 ft.) over grower's practiceplants.

FIGS. 21A-21B shows potato yields Reveille Russet Fresh Market Bakingpotatoes in Hartley County, Tex. (A) and Russet Burbank potatoes inWalworth County, Wis. (B). The soil treatment composition was applied tothe Reveille Russets once at planting, at 3 fl. oz./acre. The treatedplants showed a 31% increase in yield over grower's practice plants(FIG. 21A). The soil treatment composition was applied to the RussetBurbank potatoes 3 times at 3 fl. oz./acre each. A 5% increase wasobserved between treatment number 3 and the grower's practice plants(FIG. 21B).

FIGS. 22A-22B show potato harvest quality for Russet Burbank potatoes(22A) and yield by grade for Colomba potatoes (22B). The Russet Burbankpotatoes were treated up to 3 times with the soil treatment composition,with the 1 treatment, 2 treatment and 3 treatment groups all exhibitingan increase in number of No. 1 Potatoes (FIG. 22A). The Colomba potatoeswere treated monthly, for a total of 3 treatments. A 26% increase inGrade A potatoes was observed compared with grower's practice potatoes.

Example 14—Strawberry Field Trials

Strawberry plants in Hillsborough County, Fla. were treated with a soiltreatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”) and compared with untreated control (standardgrower's practice) plants.

Fruit diameter and weight were measured for two groups of Radiancestrawberries treated twice with the soil treatment composition at 3 fl.oz./acre (FIG. 23A). A 6% and an 8.5% increase in fruit diameter wasobserved for the treated strawberries over grower's practice plants. Avisible increase in fruit size was observed between the treated anduntreated strawberries (FIG. 23B). Furthermore, a 15% increase and a 17%increase in weight (g) was observed for the two groups compared withuntreated control groups (FIG. 23C).

Brix ratings, canopy width, root mass, fruit count, average flats/acreand total flats were also measured and compared with grower's practicestrawberry plants:

An 11% increase in degrees Brix was observed for Radiance strawberriesthat received 2 monthly treatments of the soil treatment composition at3 fl. oz./acre (FIG. 24);

a 6% increase in canopy width (in) was observed for Brilliancestrawberries that received 5 monthly treatments of the soil treatmentcomposition at 6 fl. oz./acre (FIG. 25);

a 20% increase in root mass (g/plant) was observed for Brilliancestrawberries that received two monthly treatments of the soil treatmentcomposition at 6 fl. oz./acre (FIG. 26);

a 7% increase in average fruit count was observed for Radiancestrawberries that received two monthly treatments of the soil treatmentcomposition at 3 fl. oz./acre (FIG. 27); and

a 22% increase in flats/acre was observed (FIG. 28A), and a 22% increasein total flats (FIG. 28B) was observed for Radiance strawberry plantsthat received 4 monthly treatments of the soil treatment composition at3 fl. oz./acre.

Example 15—Corn Field Trials

DKC 56-45 variety corn plants in Walworth County, Wis. were treated witha soil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”) either once at planting, once at mid-season,or twice (once at planting and once at mid-season). The treatment groupswere compared with untreated control (standard grower's practice)plants.

Root weight (g), plant vigor rating (1-5), and yield (bu/acre) weremeasured. The double-treatment group showed the greatest increase inroot weight (24%) (FIG. 29), vigor (FIG. 30), and yield (5%) (FIG. 31)compared with the grower's practice plants.

Example 16—Cotton Field Trials

Two groups of cotton plants in Lubbock County, Tex. were treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”) and were compared with untreated control(standard grower's practice) plants.

Each group received three treatments (bi-weekly) of the soil treatmentcomposition at 3 fl. oz./acre. Projected cotton yields showed a 14%increase and 10% increase in pounds of cotton per acre for the twogroups, respectively, compared with grower's practice plants (FIG. 32).

Example 17—Tobacco Field Trials

CC1063 Variety tobacco plants in Lenoir County, N.C. were treated with asoil treatment composition according to an embodiment of the subjectinvention (“Rhizolizer™”) and compared with untreated control (standardgrower's practice) plants.

Plants were treated once at the time of transplant (3 fl. oz./acre).Average chlorophyll (relative greenness), average leaf width (in) andaverage leaf length (in) were measured. A 4% increase in relativegreenness, a 35% increase in leaf width, and an 18% increase in leaflength was observed (FIG. 33).

Example 18—Peanut Field Trials

Runner peanuts in Bulloch County, Ga. were treated with a soil treatmentcomposition according to an embodiment of the subject invention(“Rhizolizer™”) and compared with untreated control (standard grower'spractice) plants.

Plants were treated once at planting (3 fl. oz./acre). Projected yields(lbs./acre) increased by 4% compared with grower's practice plants (FIG.34).

Example 19—Microbial Strains

The subject invention utilizes beneficial microbial strains. Trichodermaharzianum strains can include, but are not limited to, T-315 (ATCC20671); T-35 (ATCC 20691); 1295-7 (ATCC 20846); 1295-22 [T-22] (ATCC20847); 1295-74 (ATCC 20848); 1295-106 (ATCC 20873); T12 (ATCC 56678);WT-6 (ATCC 52443): Rifa T-77 (CMI CC 333646); T-95 (60850); T12m (ATCC20737); SK-55 (No. 13327; BP 4326 NIBH (Japan)); RR17Bc (ATCC PTA 9708);TSHTH20-1 (ATCC PTA 10317); AB 63-3 (ATCC 18647); OMZ 779 (ATCC 201359);WC 47695 (ATCC 201575); m5 (ATCC 201645); (ATCC 204065); UPM-29 (ATCC204075); T-39 (EPA 119200); and/or F11Bab (ATCC PTA 9709).

Bacillus amyloliquefaciens strains can include, but are not limited to,FZB24 (EPA 72098-5; BGSC 10A6), TA208, NJN-6, N2-4, N3-8, and thosehaving ATCC accession numbers 23842, 23844, 23843, 23845, 23350 (strainDSM 7), 27505, 31592, 49763, 53495, 700385, BAA-390, PTA-7544, PTA-7545,PTA-7546, PTA-7549, PTA-7791, PTA-5819, PTA-7542, PTA-7790, and/orPTA-7541.

We claim:
 1. A soil treatment composition for enhancing plant immunity,health, growth and/oryield, the composition comprising a firstmicroorganism and a second microorganism, and/or a growth by-product ofthe first and/or second microorganism, wherein the first microorganismand the second microorganism are both non-pathogenic, spore-formingmicroorganisms; and wherein the first microorganism is a Trichodermaspp. fungus and the second microorganism is a Bacillus spp. bacterium.2. The composition of claim 1, wherein the Trichoderma fungus isTrichoderma harzianum.
 3. The composition of claim 1, wherein theBacillus bacterium is Bacillus amyloliquefaciens.
 4. The composition ofclaim 1, wherein the composition comprises a cell count ratio of 1:4,Trichoderma to Bacillus.
 5. The composition of claim 1, furthercomprising one or more additional microorganisms selected fromMyxococcus xanthus, Azotobacter vinelandii and Frateuria aurantia.
 6. Amethod of enhancing plant immunity, health, growth and/oryield, whereinthe method comprises applying a first microorganism and/or a growthby-product thereof, and a second microorganism and/or a growthby-product thereof, to a plant and/or its surrounding environment,wherein the first microorganism is a spore-forming Trichoderma spp.fungus, and the second microorganism is a spore-forming Bacillus spp.bacterium.
 7. The method of claim 6, wherein the Trichoderma fungus isTrichoderma harzianum.
 8. The method of claim 7, wherein the Bacillusbacterium is Bacillus amyloliquefaciens.
 9. The method of claim 6,wherein a soil treatment composition comprising both the firstmicroorganism and the second microorganism, and/or one or more growthby-products thereof, is applied to the plant and/or its surroundingenvironment.
 10. The method of claim 6, wherein the first microorganism,second microorganism and/or growth by-products thereof, are applied tothe plant and/or its surrounding environment using an irrigation system.11. The method of claim 6, wherein the wherein the first microorganism,second microorganism and/or growth by-products thereof, are applied tothe plant and/or its surrounding environment alongside a source of oneor more nutrients selected from nitrogen, phosphorous, and potassium.12. The method of claim 6, wherein the first microorganism, secondmicroorganism and/or growth by-products thereof, are not appliedsimultaneously with, or within 7 to 10 days before or after, applicationof benomyl, dodecyl dimethyl ammonium chloride, hydrogendioxide/peroxyacetic acid, imazilil, propiconazole, tebuconazole, ortriflumizole to the plant and/or its surrounding environment.
 13. Themethod of claim 6, wherein the plant has compromised immune health dueto an infection from a pathogenic agent or due to an environmentalstressor.
 14. The method of claim 13, used to improve the immune healthof the plant.
 15. The method of claim 13, wherein the plant is a citrusplant affected by citrus greening disease and/or citrus canker disease.16. The method of claim 6, used to improve one or more qualities ofsoil.
 17. The method of claim 16, used to improve nutrient retention indepleted soil.
 18. The method of claim 6, used to enhance nutrientabsorption in plant roots.
 19. The method of claim 6, wherein the plantis a crop plant.
 20. The method of claim 19, wherein the crop plant isselected from citrus, tomato, sod, potato, sugarcane, grapes,watermelon, cantaloupe, lettuce, almond, onion, carrot, berries andcotton.
 21. The method of claim 6, wherein the first microorganism,second microorganism and/or growth by-products thereof, are sprayed ontothe plant and/or its surrounding environment using a handheld lawn andgarden sprayer.
 22. The method of claim 21, wherein the method is usedfor home and garden applications.
 23. A method of enhancing plantimmunity, health, growth and/or yield, comprising: applying a soiltreatment composition of claim 1 to a plant and/or its surroundingenvironment, and, optionally, applying nutrients and/or prebiotics formicrobial growth.